Anti-pd-l1 combinations for treating tumors

ABSTRACT

The present invention relates to therapeutic combinations and methods for treating cancers using combination therapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/401,843, filed Jan. 9, 2017, which is a continuation-in-part ofPCT/CN2015/083585, filed Jul. 8, 2015, which claims the benefit of, andpriority to, Chinese Patent Application Serial Nos. 201410325480.9,filed Jul. 9, 2014, and 201410440824.0, filed Sep. 1, 2014, the entiredisclosures of which are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to therapeutic combinations and methodsfor treating cancers using combination therapy.

BACKGROUND OF THE INVENTION

Therapeutic antibodies have been used in clinical applications for overtwenty years. Currently, there are fifteen anti-tumor antibody drugs inclinic, including Rituxan (1997), Herceptin (1998), Mylotarg (2000),Campath (2001), Zevalin (2002), Bexxer (2003), Avastin (2004), Erbitux(2004), Vectibix (2006), Arzerra (2009); Benlysta (2011); Yervoy (2011),Adcetris (2011), Perjeta (2012), and Kadcyla (2013). These antibodiestarget mainly four molecules: EGFR, Her2, CD20 and VEGF.

In general, therapeutic antibodies kill tumor cells via three mechanisms(Scott A M, Wolchok J D, Old L J. Antibody therapy of cancer. Nat RevCancer. (2012), 12:278-87): (1) Direct antibody action, that is,blockade or agonist activity of ligand/receptor signaling, induction ofapoptosis, and delivery of drugs or cytotoxic agents. Antibody receptoractivation activity can produce direct tumor cell killing effect. Forexample, some antibodies can bind to receptors on the surface of tumorcells, activate the receptor, leading to apoptosis (e.g., inmitochondria). Antibodies can also mediate tumor cell killing byreceptor-antagonistic activity. For example, certain antibodies can bindto cell surface receptors and block dimerization, kinase activation anddownstream signaling, thereby inhibiting proliferation and promoteapoptosis. Binding of antibodies to an enzyme can lead toneutralization, signal abrogation, and cell death. (2) Throughimmune-mediated cell killing mechanisms include complement-dependentcytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity(ADCC), T cell function regulation, etc. Immune-mediated killing oftumor cells can be accomplished through the following ways: induction ofphagocytosis, complement activation, antibody-dependent cell-mediatedcytotoxicity, genetically-modified T cells being targeted to the tumorby single-chain variable fragment (scFv), through antibody-mediatedantigenic cross presentation to dendritic cell to activate T cells,inhibition of T cell inhibitory receptors, such as cytotoxic Tlymphocyte-associated antigen 4 (CTLA4). Of them, the Fc portion of theantibody feature is especially important for CDC and ADCC-mediated tumorcell killing effect. (3) Specific effect of antibody on tumorvasculature and matrix, through trapping of vascular receptor antagonistor ligand to induce vascular and stromal cells ablation, including:stromal cell inhibition, delivery of toxins to stromal cells, anddelivery of toxins to the vasculature. (Scott A M, Wolchok J D, Old L J.Antibody therapy of cancer. Nat Rev Cancer. 2012, 12 (4):278-87).

Therapeutic monoclonal antibody drugs have advanced anti-cancer drugresearch and development. However, some issues still need further studyto be solved, such as antibody immunogenicity, tolerance of long-termuse of tumor target, and long-term effects of simple single blockade ofsignal transduction pathway. In short, a simple majority of antibodiesare difficult to achieve long-term efficient inhibition and killing oftumor cells.

Antibody-drug conjugates combine targeting function and small moleculedrug with particular pharmacokinetics. The structure of antibody-drugconjugates is the attachment of a monoclonal antibody with targetingfunction to a compound with specific pharmacological properties. Thistechnique requires the therapeutic antibody have binding specificity toa target, to be coupled to a molecule with therapeutic effect or otherfunctions such as cyto-toxins. Many factors affect the effect of thistype of antibodies, such as endocytosis of the coupled antibody,stability of the coupling, and release and killing activity of thetoxins.

Antibodies-drug conjugates have direct and indirect anti-cancer effect.The antibody blocks or activates ligand/receptor signaling, inducesapoptosis, and at the same time can present or deliver payload drugdirectly or indirectly (such as a drug, toxin, small interfering RNA orradioisotope) to the tumor cells. Therapeutic antibody drug conjugateutilizes dual characteristics of the antibody and the coupled drug,first is the binding function that it specifically binds to the targetmolecule, second is the tumor cell killing function of the antibodyitself, and the third is the particular effect of the conjugated drug.Current antibody-drug conjugates drugs are limited in how to kill tumorcells directly. However, because of the tough requirement oftechnologies in antibody, linker molecule, toxin molecules, andconjugation, as well as the limitation of bringing toxins within thetumor microenvironment molecules, there are still some difficulties inactual clinical studies.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a therapeutic combination,comprising: (i) an effective amount of a PD-L/PD-1 Axis antagonist; and(ii) an effective amount of an immunotherapeutic that is capable ofactivating a human plasmacytoid dendritic cell, myeloid dendritic cell,or NK cell, or a combination thereof.

In some embodiments, the PD-L/PD-1 Axis antagonist is selected from thegroup consisting of a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist.

In some embodiments, the PD-L/PD-1 Axis antagonist is a PD-1 bindingantagonist.

In some embodiments, the PD-1 binding antagonist inhibits the binding ofPD-1 to its ligand binding partners.

In some embodiments, the PD-1 binding antagonist inhibits the binding ofPD-1 to PD-L1.

In some embodiments, the PD-1 binding antagonist inhibits the binding ofPD-1 to PD-L2.

In some embodiments, the PD-1 binding antagonist inhibits the binding ofPD-1 to both PD-L1 and PD-L2.

In some embodiments, the PD-1 binding antagonist is an antibody, such asMDX-1106, Merck 3745, CT-011, AMP-224 or AMP-514.

In some embodiments, the PD-L/PD-1 Axis antagonist is a PD-L1 bindingantagonist.

In some embodiments, the PD-L1 binding antagonist inhibits the bindingof PD-L1 to PD-1.

In some embodiments, the PD-L1 binding antagonist inhibits the bindingof PD-L1 to B7-1.

In some embodiments, the PD-L1 binding antagonist inhibits the bindingof PD-L1 to both PD-1 and B7-1.

In some embodiments, the PD-L1 binding antagonist is an antibody, suchas one selected from the group consisting of: YW243.55.S70, MPDL3280A,MDX-1105, MEDI-4736, and MSB0010718C.

In some embodiments, the PD-L/PD-1 Axis antagonist is a PD-L2 bindingantagonist.

In some embodiments, the PD-L2 binding antagonist is an antibody.

In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

In some embodiments, the treatment results in a sustained response inthe individual after cessation of the treatment.

In some embodiments, the immunotherapeutic is administered continuously,intermittently.

In some embodiments, the immunotherapeutic is administered before thePD-L/PD-1 Axis antagonist.

In some embodiments, the immunotherapeutic is administered simultaneouswith the PD-L/PD-1 Axis antagonist.

In some embodiments, the immunotherapeutic is administered after thePD-L/PD-1 Axis antagonist.

In some embodiments, the individual has colorectal cancer, melanoma,non-small cell lung cancer, ovarian cancer, breast cancer, pancreaticcancer, a hematological malignancy or renal cell carcinoma.

In some embodiments, wherein the PD-L/PD-1 Axis antagonist isadministered intravenously, intramuscularly, subcutaneously, topically,orally, transdermally, intraperitoneally, intraorbitally, byimplantation, by inhalation, intrathecally, intraventricularly, orintranasally.

In some embodiments, the immunotherapeutic is capable of bindingspecifically to human TLR7 and/or TLR8.

In some embodiments, the immunotherapeutic comprises: (a)single-stranded RNA (ssRNA), preferably ORN02, ORN06, ssPoly(U),ssRNA40, ssRNA41, ssRNA-DR, or Poly(dT); or (b) a receptor ligandanalogs, preferably CL075, CL097, CL264, CL307, Gardiquimod, Loxoribine,Imiquimod, or Resiquimod.

In some embodiments, the immunotherapeutics is a compound of any one offormula (I) to (XIXb), or a pharmaceutically acceptable salt or solvatethereof.

In some embodiments, the immunotherapeutic has a structure of Formula(I):

wherein dashed line represents bond or absence of bond:X is S or —NR₁, R₁ is -W₀-W₁-W₂-W₃-W₄.W₀ is a bond, alkyl, alkenyl, alkynyl, alkoxy, or -alkyl-S-alkyl-,W₁ is a bond, —O—, or —NR₂—, wherein R₂ is hydrogen, alkyl or alkenyl,W₂ is a bond, —O—, —C(O)—, —C(S)—, or —S(O)₂—,W₃ is a bond, —NR₃—, wherein R₃ is hydrogen, alkyl or alkenyl,W₄ is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, aryl,aryloxy, heteroaryl, or heterocyclyl, each of which is optionallysubstituted by one or more substituents selected from the groupconsisting of hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl,aryl, heteroaryl, heterocyclyl, —NH₂, nitro, -alkyl-hydroxyl,-alkyl-aryl, -alkyl-heteroaryl, -alkyl-heterocyclyl, —O—R₄, —O-alkyl-R₄,-alkyl-O—R₄, —C(O)—R₄, -alkyl-C(O)—R₄, -alkyl-C(O)—O—R₄, —C(O)—O—R₄,—S—R₄, —S(O)₂—R₄, —NH—S(O)₂—R₄, -alkyl-S—R₄, -alkyl-S(O)₂—R₄, —NHR₄,—NR₄R₄, —NH-alkyl-R₄, halogen, —CN, —NO₂, and —SH, wherein R₄ isindependently hydrogen, alkyl, alkenyl, -alkyl-hydroxyl, aryl,heteroaryl, heterocyclyl, or haloalkyl;Z is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl, haloalkyl,heteroaryl, heterocyclyl, each of which can be optionally substituted byone or more substituents selected from the group consisting of hydroxyl,alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl,halogen, cyano, nitro, —N(R₅)₂, -alkoxy-alkyl, -alkoxy-alkenyl,—C(O)-alkyl, —C(O)—O-alkyl, —O—C(O)-alkyl, —C(O)—N(R₅)₂, aryl,heteroaryl, —CO-aryl, and —CO-heteroaryl, wherein each R₅ isindependently hydrogen, alkyl, haloalkyl, -alkyl-aryl, or-alkyl-heteroaryl;R is hydrogen, alkyl, alkoxy, haloalkyl, halogen, aryl, heteroaryl,heterocyclyl, each of which is optionally substituted by one or moresubstituents selected from the group consisting of hydroxyl, alkoxy,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl,—NH₂, nitro, -alkyl-hydroxyl, -alkyl-aryl, -alkyl-heteroaryl,-alkyl-heterocyclyl, —O—R₄, —O-alkyl-R₄, -alkyl-O—R₄, —C(O)—R₄,—C(O)—NH—R₄, —C(O)—NR₄R₄, -alkyl-C(O)—R₄, -alkyl-C(O)—O—R₄, —C(O)—O—R₄,—O—C(O)—R₄, —S—R₄, —C(O)—S—R₄, —S—C(O)—R₄, —S(O)₂—R₄, —NH—S(O)₂—R₄,-alkyl-S—R₄, -alkyl-S(O)₂—R₄, —NHR₄, —NR₄R₄, —NH-alkyl-R₄, halogen, —CN,and —SH, wherein R₄ is independently hydrogen, alkyl, alkenyl, alkoxy,-alkyl-hydroxyl, aryl, heteroaryl, heterocyclyl, or haloalkyl;n is 0, 1, 2, 3, or 4;Y is —NR₆R₇, —CR₆R₇R₈, or -alkyl-NH₂, each of which can be optionallysubstituted by one or more substituents selected from the groupconsisting of hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, —NH₂, halogen,—N(R₅)₂, -alkoxyalkyl, -alkoxy-alkenyl, —C(O)-alkyl, —C(O)—O-alkyl,—C(O)—N(R₅)₂, aryl, heteroaryl, —CO-aryl, and —CO-heteroaryl.wherein R₆, R₇ and R₈ are independently hydrogen, alkyl, alkenyl,alkoxy, alkylamino, dialkylamino, alkylthio, arylthio, -alkyl-hydroxyl,-alkyl-C(O)—O—R₉, -alkyl-C(O)—R₉, or -alkyl-O—C(O)—R₉, wherein each R₅is independently hydrogen, alkyl, haloalkyl, -alkyl-aryl, or-alkyl-heteroaryl, wherein R₉ is hydrogen, alkyl, alkenyl, halogen, orhaloalkyl; andX and Z taken together may optionally form a (5-9)-membered ring.

In some embodiments, the immunotherapeutic is a compound selected fromthe group consisting of: 2-propylthiazolo[4,5-c]quinolin-4-amine,1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine,4-amino-2-(ethoxymethyl)-a,a-di-methyl-1H-imidazo[4,5-c]quinoline-1-ethanol,1-(4-amino-2-ethylaminomethylimidazo-[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol,N-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl-]methanesulfonamide,4-amino-2-ethoxymethyl-aa-dimethyl-6,7,8,9-tetrahydro-1h-imidazo[4,5-c]quinoline-1-ethanol,4-amino-aa-dimethyl-2-methoxyethyl-1h-imidazo[4,5-c]quinoline-1-ethanol,1-{2-[3-(benzyloxy)propoxy]ethyl}-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-amine,N-[4-(4-amino-2-butyl-1H-imidazo[4,5-c][1,5]naphthyridin-1-yl)butyl]-n′-butylurea,N1-[2-(4-amino-2-butyl-1H-imidazo[4,5-c][1,5]naphthyridin-1-yl)ethyl]-2-amino-4-methylpentanamide,N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy}ethyl)-n′-phenylurea,1-(2-amino-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-amine,1-{4-[(3,5-dichlorophenyl)sulfonyl]butyl}-2-ethyl-1H-imidazo[4,5-c]quinolin-4-amine,N-(2-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy}ethyl)-n′-cyclohexylurea,N-{3-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]propyl}-n′-(3-cyanophenyl)thiourea,N-[3-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)-2,2-dimethylpropyl]benzamide,2-butyl-1-[3-(methylsulfonyl)propyl]-1H-imidazo[4,5-c]quinolin-4-amine,N-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}-2-ethoxyacetamide,1-[4-amino-2-ethoxymethyl-7-(pyridin-4-yl)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol,1-[4-amino-2-(ethoxymethyl)-7-(pyridin-3-yl)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol,N-{3-[4-amino-1-(2-hydroxy-2-methylpropyl)-2-(methoxyethyl)-1H-imidazo[4,5-c]quinolin-7-yl]phenyl}methanesulfonamide,1-[4-amino-7-(5-hydroxymethylpyridin-3-yl)-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol,3-[4-amino-2-(ethoxymethyl)-7-(pyridin-3-yl)-1H-imidazo[4,5-c]quinolin-1-yl]propane-1,2-diol,1-[2-(4-amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-1-yl)-1,1-dimethylethyl]-3-propylurea,1-[2-(4-amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-1-yl)-1,1-dimethylethyl]-3-cyclopentylurea,1-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]-2-(ethoxymethyl)-7-(4-hydroxymethylphenyl)-1H-imidazo[4,5-c]quinolin-4-amine,4-[4-amino-2-ethoxymethyl-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-7-yl]-N-methoxy-N-methylbenzamide,2-ethoxymethyl-N1-isopropyl-6,7,8,9-tetrahydro-1H-imidazo[4,5-c]quinoline-1,4-diamine,1-[4-amino-2-ethyl-7-(pyridin-4-yl)-1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol,N-[4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide,andN-[4-(4-amino-2-butyl-1H-imidazo[4,5-c][1,5]naphthyridin-1-yl)butyl]-n′-cyclohexylurea.

In some embodiments, the immunotherapeutic has a structure of Formula(II):

wherein V is —NR₆R₇, wherein each of R₆ and R₇ is independentlyhydrogen, alkyl, alkenyl, alkoxy, alkylamino, dialkylamino, alkylthio,arylthio, -alkyl-hydroxyl, -alkyl-C(O)—O—R₉, -alkyl-C(O)—R₉, or-alkyl-O—C(O)—R₉, wherein R₉ is hydrogen, alkyl, alkenyl, halogen, orhaloalkyl;R₁₀ and R₁₁ are independently hydrogen, alkyl, alkenyl, aryl, haloalkyl,heteroaryl, heterocyclyl, or cycloalkyl, each of which is optionallysubstituted by one or more substituents selected from the groupconsisting of hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, halogen,—N(R₅)₂, -alkoxy-alkyl, -alkoxy-alkenyl, —C(O)-alkyl, —C(O)—O-alkyl,—C(O)—N(R₅)₂, aryl, heteroaryl, —CO-aryl, and —CO-heteroaryl, whereineach R₅ is independently hydrogen, alkyl, haloalkyl, -alkyl-aryl, or-alkyl-heteroaryl.

In some embodiments, the therapeutic combination or pharmaceuticalcomposition of the present invention further comprises an effectiveamount of an additional therapeutic agent, such as an anticancer agent.

In some embodiments, the anticancer agent is an antimetabolite, aninhibitor of topoisomerase I and II, an alkylating agent, a microtubuleinhibitor, an antiandrogen agent, a GNRh modulator or mixtures thereof.

In some embodiments, the additional therapeutic agent is achemotherapeutic agent selected from the group consisting of tamoxifen,raloxifene, anastrozole, exemestane, letrozole, imatanib, paclitaxel,cyclophosphamide, lovastatin, minosine, gemcitabine, cytarabine,5-fluorouracil, methotrexate, docetaxel, goserelin, vincristine,vinblastine, nocodazole, teniposide etoposide, gemcitabine, epothilone,vinorelbine, camptothecin, daunorubicin, actinomycin D, mitoxantrone,acridine, doxorubicin, epirubicin, or idarubicin.

In another aspect, the present invention provides a method for treatinga disease condition in a subject that is in need of such treatment,comprising administering to the subject the therapeutic combination orpharmaceutical composition provided herein.

In some embodiments, the diseases condition is tumor. In someembodiments, the disease condition comprises abnormal cellproliferation.

In some embodiments, the abnormal cell proliferation comprises apre-cancerous lesion. In some embodiments, the abnormal proliferation isof cancer cells.

In some embodiments, the cancer is selected from the group consistingof: breast cancer, colorectal cancer, diffuse large B-cell lymphoma,endometrial cancer, follicular lymphoma, gastric cancer, glioblastoma,head and neck cancer, hepatocellular cancer, lung cancer, melanoma,multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer,and renal cell carcinoma.

In some embodiments, the immunotherapeutic is of an amount that iscapable of:

-   -   (1) inducing IFN-α in an enriched human blood DCs;    -   (2) inducing TNF-α in an enriched human blood DCs;    -   (3) inducing IL-12-α in an enriched human blood DCs;    -   (4) activating CD45+ immune cells in tumor microenvironment;    -   (5) activating CD4+ and CD8+ T cells in tumor microenvironment;    -   (6) activating NK cells in tumor microenvironment;    -   (7) activating plasmacytoid dendritic cells (pDC) and myeloid        dendritic cells (mDc) in tumor microenvironment;    -   (8) activating macrophages and Monocytes in tumor        microenvironment; and/or    -   (9) increasing migratory DCs in draining lymph nodes.

In some embodiments, the method comprises administering to the subjectan oral formulation comprising the immunotherapeutic (such as R848 andits analogues) in a dose of between about 0.0005 mg/kg, 0.0006 mg/kg,0.0007 mg/kg, 0.0008 mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0.002 mg/kg,0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008mg/kg, 0.009 mg/kg, or 0.01 mg/kg, to about 0.02 mg/kg, twice per week.

In some embodiments, the method comprises administering to the subjectan oral formulation comprising the immunotherapeutic (such as R848 andits analogues) in a dose of less than or about 0.005 mg/kg, 0.006 mg/kg,0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, or 0.01 mg/kg, twice per week.

In some embodiments, the method comprises administering to said subjectan intravenous formulation comprising said immunotherapeutic (such asR848 and its analogues) in a dose of between about 0.0005 mg/kg, 0.0006mg/kg, 0.0007 mg/kg, 0.0008 mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0,002mg/kg, 0,003 mg/kg, 0,004 mg/kg, 0,005 mg/kg, or 0.006 mg/kg to about0.015 mg/kg, weekly. In some embodiments, the method comprisesadministering to said subject an intravenous formulation comprising saidimmunotherapeutic (such as R848 and its analogues) in a dose of betweenabout 0.0008 mg/kg to about 0.0067 mg/kg, weekly.

In some embodiments, the method comprises administering to said subjectan intravenous formulation comprising said immunotherapeutic in a doseof less than or about 0,003 mg/kg, 0,004 mg/kg, 0,005 mg/kg, or 0.006mg/kg to about 0.007 mg/kg, weekly.

In some embodiments, the immunotherapeutic in the subject has a localconcentration that is between about 0.005 μg/ml to about 12 μg/ml.

In some embodiments, the immunotherapeutic in the subject has a localconcentration that is is between about 0.05 μg/ml, 0.1 μg/ml, 0.15μg/ml, 0.2 μg/ml, 0.3 μg/ml, or 0.4 μg/ml, to about 0.5 μg/ml.

In a further aspect, the present invention provides a kit that containsthe therapeutic combination provided herein, and optionally with aninstruction.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts effects of anti-PDL1 mAb treatment on SCCVII tumourgrowth. SCCVII Tumours were inoculated as described in the Materials andmethods. Tumour-inoculated mice received an intraperitoneal injection ofcontrol rat immunoglobulin or anti-PDL1 mAb (200 ug/mouse) combinationwith TLRL three times a week. The mean tumour volume±SD was determinedin each group of five to eight mice.

FIG. 2 depicts effects of anti-PDL1 mAb treatment on CT26 tumour growth.CT26 Tumours were inoculated as described in the Materials and methods.Tumour-inoculated mice received an intraperitoneal injection of controlrat immunoglobulin or anti-PDL1 mAb (200 ug/mouse) combination with TLRLthree times a week. The mean tumour volume±SD was determined in eachgroup of five to eight mice.

FIGS. 3A-3G depict analysis of cytokine production by enriched human DCsfrom three healthy donors. Enriched human DCs were plated in a 96-wellplate and cultured with allogeneic untreated (medium) or treateddifferent concentration of TLRL directly for 20-22 h in 37° C.incubator. The supernatants were collected and human IFN-α, IL-12(p70)and TNF-α were analyzed by ELISA. Data are given as mean±SD oftriplicate cultures. Three independent experiments from three healthydonors were performed. A: TLRL induced IFN-α expression in enrichedhuman blood DCs (CD3+/CD19+/CD14+/CD16+) from donor 1. B: TLRL inducedIFN-α expression in enriched human blood DCs in Experiment #2 from donor2. C: TLRL induced TNF-α expression in enriched human blood DCs inExperiment #2 from donor 2. D: TLRL induced IL-12 expression in enrichedhuman blood DCs in Experiment #2 from donor 2. E: TLRL induced IFN-αexpression in enriched human blood DCs Experiment #3 from donor 3. F:TLRL induced TNF-α expression in enriched human blood DCs in Experiment#3 from donor 3. G: TLRL induced IL-12 expression in enriched humanblood DCs in Experiment #3 from donor 3.

FIGS. 4A-C depicts expression of IFN inducible genes in mouse PBMC afterTLRL injection. RNA was isolated from PBMCs cryopreserved with TRIzolreagent at variable time points and Relative expression of IFN induciblegenes were determined by quantitative RT-PCR. MX2 gene was detected overtime course of 5 hours post TLRL injection (4A) and MX2 and ISG15 geneswere measured with various dose of TLRL at 2 hours post injection (4B)and (4C), respectively. Values indicate the mRNA expression of indicatedIFN inducible genes relative to housekeeping gene Actin. Bar graphsrepresent data from 3 individual animals. **P<0.01; ***P<0.001.

FIG. 5 depicts tumor volumes of mice treated with anti-PD1 alone. R848alone, and combination of cnti-PD1 and R848 against couse colon cellsC26 in BALB/c mice. Tumor volumes are shown as the mean+SEM of 10 miceper group. Mice treated with the combination of ANTI-PD-1 and R848intravenously had the best antitumor response with 96.15% (p<0.01) tumorinhibition.

FIG. 6 depicts Tumor columes of mice treated with anti-PD-L1 alone, R848alone, and combination of anti-PD-L1 and R848 against aouse colon cellsC26 in BALB/c mice. Tumor volumes are shown as the mean+SEM of 10 miceper group. Mice treated with the combination of Anti-PD-L1 and R848intravenously had the best antitumor response with 96.15% (p<0.01) tumorinhibition.

DETAILED DESCRIPTION OF THE INVENTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the illustratedordering of acts or events, as some acts may occur in different ordersand/or concurrently with other acts or events.

Furthermore, not all illustrated acts or events are required toimplement a methodology in accordance with the present invention.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10% more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

I. Definitions and Abbreviations

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization are those well-known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences, which are provided throughout this document. Thenomenclature used herein and the laboratory procedures in analyticalchemistry, and organic synthetic described below are diose well-knownand commonly employed in the art. Standard techniques, or modificationsthereof, are used for chemical syntheses and chemical analyses.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups, whichare limited to hydrocarbon groups, are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

In general, an “acyl substituent” is also selected from the group setforth above. As used herein, the term “acyl substituent” refers togroups attached to, and fulfilling the valence of a carbonyl carbon thatis either directly or indirectly attached to the polycyclic nucleus ofthe compounds of the present invention.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

As used herein, the term “haloalkyl” refers to an alkyl as definedherein, that is substituted by one or more halo groups as definedherein. Preferably the haloalkyl can be monohaloalkyl, dihaloalkyl orpolyhaloalkyl including perhaloalkyl. A monohaloalkyl can have one iodo,bromo, chloro or fluoro within the alkyl group. Dihaloalkyl andpolyhaloalkyl groups can have two or more of the same halo atoms or acombination of different halo groups within the alkyl. Preferably, thepolyhaloalkyl contains up to 12, 10, or 8, or 6, or 4, or 3, or 2 halogroups. Non-limiting examples of haloalkyl include fluoromethyl,difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl,trichloromethyl, pentafluoroethyl, heptafluoropropyl,difluorochloromethyl, dichlorofluoromethyl, difluoroethyl,difluoropropyl, dichloroethyl and dichloropropyl. A perhaloalkyl refersto an alkyl having all hydrogen atoms replaced with halo atoms.

As used herein, the term “heteroaryl” refers to a 5-14 memberedmonocyclic- or bicyclic- or fused polycyclic-ring system, having 1 to 8heteroatoms selected from N, O, S or Se. Preferably, the heteroaryl is a5-10 membered ring system. Typical heteroaryl groups include 2- or3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl, 3-,4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl,2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-1,2,4-triazolyl,4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-pyrazinyl, 2-, 4-, or5-pyrimidinyl.

The term “heteroaryl” also refers to a group in which a heteroaromaticring is fused to one or more aryl, cycloaliphatic, or heterocycloalkylrings, where the radical or point of attachment is on the heteroaromaticring. Nonlimiting examples include but are not limited to 1-, 2-, 3-,5-, 6-, 7-, or 8-indolizinyl, 1-, 3-, 4-, 5-, 6-, or 7-isoindolyl, 2-,3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-indazolyl, 2-,4-, 5-, 6-, 7-, or 8-purinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, or9-quinolizinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinoliyl, 1-, 3-, 4-, 5-,6-, 7-, or 8-isoquinoliyl, 1-, 4-, 5-, 6-, 7-, or 8-phthalazinyl, 2-,3-, 4-, 5-, or 6-naphthyridinyl, 2-, 3-, 5-, 6-, 7-, or 8-quinazolinyl,3-, 4-, 5-, 6-, 7-, or 8-cinnolinyl, 2-, 4-, 6-, or 7-pteridinyl, 1-,2-, 3-, 4-, 5-, 6-, 7-, or 8-4aH carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-,or 8-carbzaolyl, 1-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-carbolinyl, 1-, 2-,3-, 4-, 6-, 7-, 8-, 9-, or 10-phenanthridinyl, 1-, 2-, 3-, 4-, 5-, 6-,7-, 8-, or 9-acridinyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9-perimidinyl,2-, 3-, 4-, 5-, 6-, 8-, 9-, or 10-phenathrolinyl, 1-, 2-, 3-, 4-, 6-,7-, 8-, or 9-phenazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or10-phenothiazinyl, 1-, 2-, 3-, 4- , 6-, 7-, 8-, 9-, or 10-phenoxazinyl,2-, 3-, 4-, 5-, 6-, or 1-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or10-benzisoqinolinyl, 2-, 3-, 4-, or 5-thieno[2,3-b]furanyl, 2-, 3-, 5-,6-, 7-, 8-, 9-, 10-, or 11-7H-pyrazino[2,3-c]carbazolyl, 2-, 3-, 5-, 6-,or 7-2H-furo[3,2-b]-pyranyl, 2-, 3-, 4-, 5-, 7-, or8-5H-pyrido[2,3-d]-o-oxazinyl, 1-, 3-, or 5-1H-pyrazolo[4,3-d]-oxazolyl,2-, 4-, or 54H-imidazo[4,5-d] thiazolyl, 3-, 5-, or8-pyrazino[2,3-d]pyridazinyl, 2-, 3-, 5-, or 6-imidazoyl-[2,1-b]thiazolyl, 1-, 3-, 6-, 7-, 8-, or 9-furo[3,4-c]cinnolinyl, 1-, 2-, 3-,4-, 5-, 6-, 8-, 9-, 10, or 1 l-4H-pyrido[2,3-c]carbazolyl, 2-, 3-, 6-,or 7-imidazo[1,2-b][1,2,4]triazinyl, 7-benzo[b]thienyl, 2-, 4-, 5-, 6-,or 7-benzoxazolyl, 2-, 4-, 5-, 6-, or 7-benzimidazolyl, 2-, 4-, 4-, 5-,6-, or 7-benzothiazolyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9-benzoxapinyl,2-, 4-, 5-, 6-, 7-, or 8-benzoxazinyl, 1-, 2-, 3-, 5-, 6-, 7-, 8-, 9-,10-, or 11-1H-pyrrolo[1,2-b][2]benzazapinyl. Typical fused heteroarylgroups include, but are not limited to 2-, 3-, 4-, 5-, 6-, 7-, or8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolinyl, 2-, 3-, 4-,5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-benzo[b]thienyl, 2-, 4-,5-, 6-, or 7-benzoxazolyl, 2-, 4-, 5-, 6-, or 7-benzimidazolyl, 2-, 4-,5-, 6-, or 7-benzothiazolyl.

As used herein, the term “heterocyclyl” or “heterocyclo” refers to anoptionally substituted, fully saturated or unsaturated, aromatic ornonaromatic cyclic group, e.g., which is a 4- to 7-membered monocyclic,7- to 12-membered bicyclic or 10- to 15-membered tricyclic ring system,which has at least one heteroatom in at least one carbon atom-containingring. Each ring of the heterocyclic group containing a heteroatom mayhave 1, 2 or 3 heteroatoms selected from nitrogen atoms, oxygen atomsand sulfur atoms, where the nitrogen and sulfur heteroatoms may alsooptionally be oxidized. The heterocyclic group may be attached at aheteroatom or a carbon atom.

Exemplary monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl,pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl,imidazolidinyl, triazolyl, oxazolyl, oxazolidinyl, isoxazolinyl,isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl,piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, 4-piperidonyl, pyridyl,pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, morpholinyl,thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone,1,3-dioxolane and tetrahydro-1,1-dioxothienyl,1,1,4-trioxo-1,2,5-thiadiazolidin-2-yl and the like.

Exemplary bicyclic heterocyclic groups include indolyl, dihydroidolyl,benzothiazolyl, benzoxazinyl, benzoxazolyl, benzothienyl,benzothiazinyl, quinuclidinyl, quinolinyl, tetrahydroquinolinyl,decahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl,decahydroisoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl,benzofuryl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl,quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such asfuro[2,3-c]pyridinyl, furo[3,2-b]-pyridinyl] or furo[2,3-b]pyridinyl),dihydroisoindolyl, 1,3-dioxo-1,3-dihydroisoindol-2-yl,dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl),phthalazinyl and the like.

Exemplary tricyclic heterocyclic groups include carbazolyl,dibenzoazepinyl, dithienoazepinyl, benzindolyl, phenanthrolinyl,acridinyl, phenanthridinyl, phenoxazinyl, phenothiazinyl, xanthenyl,carbolinyl and the like.

The term “heterocyclyl” further refers to heterocyclic groups as definedherein substituted with 1, 2 or 3 substituents selected from the groupsconsisting of the following:

(a) alkyl;

(b) hydroxy (or protected hydroxy);

(c) halo;

(d) oxo, i.e., ═O;

(e) amino, alkylamino or dialkylamino;

(f) alkoxy;

(g) cycloalkyl;

(h) carboxy;

(i) heterocyclooxy, wherein heterocyclooxy denotes a heterocyclic groupbonded through an oxygen bridge;

(j) alkyl-O—C(O)—;

(k) mercapto;

(l) nitro;

(m) cyano;

(n) sulfamoyl or sulfonamido;

(o) aryl;

(p) alkyl-C(O)—O—;

(q) aryl-C(O)—O—;

(r) aryl-S—;

(s) aryloxy;

(t) alkyl-S—;

(u) formyl, i.e., HC(O)—;

(v) carbamoyl;

(w) aryl-alkyl-; and

(x) aryl substituted with alkyl, cycloalkyl, alkoxy, hydroxy, amino,alkyl-C(O)—NH—, alkylamino, dialkylamino or halogen.

As used herein, the term “alkenyl” refers to a straight or branchedhydrocarbon group having 2 to 20 carbon atoms and that contains at leastone double bonds. The alkenyl groups preferably have about 2 to 8 carbonatoms.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings), which are fused togetheror linked covalently. The term “heteroaryl” refers to aryl groups (orrings) that contain from one to four heteroatoms selected from N, O, andS, wherein the nitrogen and sulfur atoms are optionally-oxidized, andthe nitrogen atom(s) are optionally quaternized. A heteroaryl group canbe attached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including diosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl.” “aryl” and“heteroaryl”) include both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl, and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generally referred to as “alkyl substituents”and “heteroakyl substituents,” respectively, and they can be one or moreof a variety of groups selected from, but not limited to: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R′R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R″′, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂ in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R′, R″, R′″ andR″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., arylsubstituted with 1-3 halogens, substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R″is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical, the arylsubstituents and heteroaryl substituents are generally referred to as“aryl substituents” and “heteroaryl substituents,” respectively and arevaried and selected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R″, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR*—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R′R″ and R″″ groupswhen more than one of these groups is present.

Two of the aryl substituents on adjacent atoms of the aryl or heteroarylring may optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)-U-, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)-B-, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d), where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆) akyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P) and silicon (Si).

As used herein, the term “aryloxy” refers to both an —O-aryl and an—O-heteroaryl group, wherein aryl and heteroaryl are defined herein.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the biological effectiveness and properties of thecompounds of this invention and, which are not biologically or otherwiseundesirable. In many cases, the compounds of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto (e.g., phenol orhydroxyamic acid). Pharmaceutically acceptable acid addition salts canbe formed with inorganic acids and organic acids. Inorganic acids fromwhich salts can be derived include, for example, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike. Organic acids from which salts can be derived include, forexample, acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like. Pharmaceutically acceptable base additionsalts can be formed with inorganic and organic bases. Inorganic basesfrom which salts can be derived include, for example, sodium, potassium,lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese,aluminum, and the like; particularly preferred are the ammonium,potassium, sodium, calcium and magnesium salts.

Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like, specifically such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. The pharmaceutically acceptable salts of the presentinvention can be synthesized from a parent compound, a basic or acidicmoiety, by conventional chemical methods. Generally, such salts can beprepared by reacting free acid forms of these compounds with astoichiometric amount of the appropriate base (such as Na, Ca, Mg, or Khydroxide, carbonate, bicarbonate, or the like), or by reacting freebase forms of these compounds with a stoichiometric amount of theappropriate acid. Such reactions are typically carried out in water orin an organic solvent, or in a mixture of the two. Generally,non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, oracetonitrile are preferred, where practicable. Lists of additionalsuitable salts can be found, e.g., in Remington's PharmaceuticalSciences, 20th ed., Mack Publishing Company, Easton, Pa., (1985), whichis herein incorporated by reference.

As used herein, the term “pharmaceutically acceptable carrier/excipient”includes any and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts, drugs, drugstabilizers, binders, excipients, disintegration agents, lubricants,sweetening agents, flavoring agents, dyes, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.Mack Printing Company, 1990, pp. 1289-1329, incorporated herein byreference). Except in so far as any conventional carrier is incompatiblewith the active ingredient, its use in the therapeutic or pharmaceuticalcompositions is contemplated.

As used herein, the term “subject” refers to an animal. Preferably, theanimal is a mammal. A subject also refers to for example, primates(e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats,mice, fish, birds and the like. In a preferred embodiment, the subjectis a human.

As used herein, the term “therapeutic combination” or “combination”refers to a combination of one or more active drug substances, i.e.,compounds having a therapeutic utility. Typically, each such compound inthe therapeutic combinations of the present invention will be present ina pharmaceutical composition comprising that compound and apharmaceutically acceptable carrier. The compounds in a therapeuticcombination of the present invention may be administered simultaneouslyor separately, as part of a regimen.

II. Compositions

In general, the present invention provides therapeutic combinations,pharmaceutical compostions, and methods for treating cancers usingcombination therapy. More specifically, the combination of immunotherapy(such as using Toll-like Receptor Ligand “TLRL” to activate DCs ininnate immunity and link to adaptive immunity) and targeted therapy(such as a PD-L/PD-1 Axis antagonist) are used to treats cancers such asgastric cancer and lung cancers.

In one aspect, the present invention provides therapeutic combinations,or pharmaceutical compositions, comprising: (i) an effective amount of aan effective amount of a PD-L/PD-1 Axis antagonist; (ii) an effectiveamount of an immunotherapeutic that is capable of activating a humandendritic cell, NK cell. Monocyte, Macrophage or tumor cell, or acombination thereof; and optionally (iii) one or more pharmaceuticallyacceptable carriers.

A therapeutic combination may be provided in a single pharmaceuticalcomposition so that both the targeted therapeutics and theimmunotherapeutic can be administered together. In alternativeembodiments, a therapeutic combination may be provided using more thanone pharmaceutical composition. In such embodiments, a targetedtherapeutic may be provided in one pharmaceutical composition and animmunotherapeutic may be provided in a second pharmaceutical compositionso that the two compounds can be administered separately such as, forexample, at different times, by different routes of administration, andthe like. Thus, it also may be possible to provide the targetedtherapeutic and the immunotherapeutic in different dosing regimens.

Unless otherwise indicated, reference to a compound can include thecompound in any pharmaceutically acceptable form, including any isomer(e.g., diastereomer or enantiomer), salt, solvate, polymorph, and thelike. In particular, if a compound is optically active, reference to thecompound can include each of the compound's enantiomers as well asracemic mixtures of the enantiomers.

In general, the targeted therapeutics and the immunotherapeutics are notlinked to each other, such as by a covalent linker.

A. PD-L/PD-1 Axis Antagonists

In general, the combination provided herein comprises an entity, such asa PD-L/PD-1 Axis antagonist that is capable of specifically binding to aparticular target, such as PD-L1, PD-L2 or PD-1. The entity is capableof binding to PD-L1, PD-L2, or PD-1 specifically or preferably incomparison to a non-target.

By “specifically binds” or “preferably binds” herein is meant that thebinding between two binding partners (e.g., between a targeting moietyand its binding partner) is selective for the two binding partners andcan be discriminated from unwanted or non-specific interactions. Forexample, the ability of an antigen-binding moiety to bind to a specificantigenic determinant can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. surface plasmon resonance technique (analyzed on aBIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), andtraditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Theterms “anti-[antigen] antibody” and “an antibody that binds to[antigen]” refer to an antibody that is capable of binding therespective antigen with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting theantigen. In some embodiments, the extent of binding of an anti-[antigen]antibody to an unrelated protein is less than about 10% of the bindingof the antibody to the antigen as measured, e.g., by a radioimmunoassay(RIA). In some embodiments, an antibody that binds to [antigen] has adissociation constant (KD) of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM,<0.01 nM, or <0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³M, e.g., from 10⁻⁹ M to 10⁻¹³ M). It is understood that the abovedefinition is also applicable to antigen-binding moieties that bind toan antigen.

By “PD-L/PD-1 Axis antagonist” herein is meant is a molecule thatinhibits the interaction of a PD-L/PD-1 axis binding partner with eitherone or more of its binding partner, so as to remove T-cell dysfunctionresulting from signaling on the PD-L/PD-1 signaling axis—with a resultbeing to restore or enhance T-cell function {e.g., proliferation,cytokine production, target cell killing). As used herein, a PD-L/PD-1Axis antagonist includes a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist.

By “PD-1 binding antagonists” herein is meant is a molecule thatdecreases, blocks, inhibits, abrogates or interferes with signaltransduction resulting from the interaction of PD-1 with one or more ofits binding partners, such as PD-L1, PD-L2. In some embodiments, thePD-1 binding antagonist is a molecule that inhibits the binding of PD-1to its binding partners. In a specific aspect, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. Forexample, PD-1 binding antagonists include anti-PD-1 antibodies, antigenbinding fragments thereof, immunoadhesins, fusion proteins,oligopeptides and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1binding antagonist reduces the negative co-stimulatory signal mediatedby or through cell surface proteins expressed on T lymphocytes mediatedsignaling through PD-1 so as render a dysfunctional T-cell lessdysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, the PD-1 binding antagonist is ananti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist isMDX-1 106 described herein. In another specific aspect, a PD-1 bindingantagonist is Merck 3745 described herein. In another specific aspect, aPD-1 binding antagonist is CT-01 1 described herein.

By “PD-L1 binding antagonists” herein is meant a molecule thatdecreases, blocks, inhibits, abrogates or interferes with signaltransduction resulting from the interaction of PD-L1 with either one ormore of its binding partners, such as PD-1, B7-1. In some embodiments, aPD-L1 binding antagonist is a molecule that inhibits the binding ofPD-L1 to its binding partners. In a specific aspect, the PD-L1 bindingantagonist inhibits binding of PD-L1 to PD-1 and/or B7-1. In someembodiments, the PD-L1 binding antagonists include anti-PD-L1antibodies, antigen binding fragments thereof, immunoadhesins, fusionproteins, oligopeptides and other molecules that decrease, block,inhibit, abrogate or interfere with signal transduction resulting fromthe interaction of PD-L1 with one or more of its binding partners, suchas PD-1, B7-1. In one embodiment, a PD-L1 binding antagonist reduces thenegative co-stimulatory signal mediated by or through cell surfaceproteins expressed on T lymphocytes mediated signaling through PD-L1 soas to render a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L1binding antagonist is an anti-PD-L1 antibody. In a specific aspect, ananti-PD-L1 antibody is YW243.55.S70 described herein. In anotherspecific aspect, an anti-PD-L1 antibody is MDX-1105 described herein. Instill another specific aspect, an anti-PD-L1 antibody is MPDL3280Adescribed herein.

By “PD-L2 binding antagonists” herein is meant a molecule thatdecreases, blocks, inhibits, abrogates or interferes with signaltransduction resulting from the interaction of PD-L2 with either one ormore of its binding partners, such as PD-1. In some embodiments, a PD-L2binding antagonist is a molecule that inhibits the binding of PD-L2 toits binding partners. In a specific aspect, the PD-L2 binding antagonistinhibits binding of PD-L2 to PD-1. In some embodiments, the PD-L2antagonists include anti-PD-L2 antibodies, antigen binding fragmentsthereof, immunoadhesins, fusion proteins, oligopeptides and othermolecules that decrease, block, inhibit, abrogate or interfere withsignal transduction resulting from the interaction of PD-L2 with eitherone or more of its binding partners, such as PD-1. In one embodiment, aPD-L2 binding antagonist reduces the negative co-stimulatory signalmediated by or through cell surface proteins expressed on T lymphocytesmediated signaling through PD-L2 so as render a dysfunctional T-cellless dysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, a PD-L2 binding antagonist is animmunoadhesin.

Antibodies

In some embodiments, the targeted therapeutic comprises an antibody, ora functional fragment thereof.

By immunoglobulin” or “antibody” herein is meant a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment. Anantibody or antibody fragment may be conjugated or otherwise derivatizedwithin the scope of the claimed subject matter. Such antibodies includeIgG1, IgG2a, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity and comprise an Fc region or aregion equivalent to the Fc region of an immunoglobulin The terms“full-length antibody”, “intact antibody”, “and “whole antibody” areused herein interchangeably to refer to an antibody having a structuresubstantially similar to a native antibody structure or having heavychains that contain an Fc region as defined herein.

By “native antibodies” herein is meant naturally occurringimmunoglobulin molecules with varying structures. For example, nativeIgG antibodies are heterotetrameric glycoproteins of about 150,000daltons, composed of two identical light chains and two identical heavychains that are disulfide-bonded. From N- to C-terminus, each heavychain has a variable region (VH), also called a variable heavy domain ora heavy chain variable domain, followed by three constant domains (CHI,CH2, and CH3), also called a heavy chain constant region. Similarly,from N- to C-terminus, each light chain has a variable region (VL), alsocalled a variable light domain or a light chain variable domain,followed by a constant light (CL) domain, also called a light chainconstant region. The light chain of an antibody may be assigned to oneof two types, called kappa (κ) and lambda (λ), based on the amino acidsequence of its constant domain.

By “antibody fragment” herein is meant a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab. Fab′, Fab′-SH,F(ab′)2, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), single-domain antibodies, and multispecific antibodiesformed from antibody fragments. For a review of certain antibodyfragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review ofscFv fragments, see e.g. Pliickthun, in The Pharmacology of MonoclonalAntibodies, vol. 113. Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragmentscomprising salvage receptor binding epitope residues and havingincreased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies areantibody fragments with two antigen-binding sites that may be bivalentor bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson etal., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad SciUSA 90, 6444-6448 (1993). Triabodies and tetrabodies are also describedin Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodiesare antibody fragments comprising all or a portion of the heavy chainvariable domain or all or a portion of the light chain variable domainof an antibody. In certain embodiments, a single-domain antibody is ahuman single-domain antibody (Domantis, Inc., Waltham, Mass.; see e.g.U.S. Pat. No. 6,248,516 B1). Antibody fragments can be made by varioustechniques, including but not limited to proteolytic digestion of anintact antibody as well as production by recombinant host cells (e.g. E.coli or phage), as described herein.

By “antigen binding domain” herein is meant the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Particularly, an antigen binding domain comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

By “variable region” or “variable domain” herein is meant the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).A single VH or VL domain may be sufficient to confer antigen-bindingspecificity.

By “hypervariable region” or “HVR” herein is meant each of the regionsof an antibody variable domain which are hypervariable in sequenceand/or form structurally defined loops ““hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as “complementarity determining regions” (CDRs),and these terms are used herein interchangeably in reference to portionsof the variable region that form the antigen binding regions. Thisparticular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, Sequences of Proteins of ImmunologicalInterest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987),where the definitions include overlapping or subsets of amino acidresidues when compared against each other. Nevertheless, application ofeither definition to refer to a CDR of an antibody or variants thereofis intended to be within the scope of the term as defined and usedherein. The exact residue numbers which encompass a particular CDR willvary depending on the sequence and size of the CDR. Those skilled in theart can routinely determine which residues comprise a particular CDRgiven the variable region amino acid sequence of the antibody.

The antibody of the present invention can be chimeric antibodies,humanized antibodies, human antibodies, or antibody fusion proteins.

By “chimeric antibody” herein is meant a recombinant protein thatcontains the variable domains of both the heavy and light antibodychains, including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, morepreferably a murine antibody, while the constant domains of the antibodymolecule are derived from those of a human antibody. For veterinaryapplications, the constant domains of the chimeric antibody may bederived from that of other species, such as a subhuman primate, cat ordog.

By “humanized antibody” herein is meant a recombinant protein in whichthe CDRs from an antibody from one species; e.g., a rodent antibody, aretransferred from the heavy and light variable chains of the rodentantibody into human heavy and light variable domains. The constantdomains of die antibody molecule are derived from those of a humanantibody. In some embodiments, specific residues of the framework regionof the humanized antibody, particularly those that are touching or closeto the CDR sequences, may be modified, for example replaced with thecorresponding residues from the original rodent, subhuman primate, orother antibody.

By “human antibody” herein is meant an antibody obtained, for example,from transgenic mice that have been “engineered” to produce specifichuman antibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain locus are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy chain and light chain loci.The transgenic mice can synthesize human antibodies specific for humanantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al. Nature Genet. 7: 13 (1994), Lonberg et al,Nature 368:856 (1994), and Taylor et al, Int. Immun. 6:579 (1994). Afully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al, Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993). Human antibodies may also begenerated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610and 5,229,275, which are incorporated herein by reference in theirentirety.

By “antibody fusion protein” herein is meant a recombinantly-producedantigen-binding molecule in which two or more of the same or differentnatural antibody, single-chain antibody or antibody fragment segmentswith the same or different specificities are linked. A fusion proteincomprises at least one specific binding site. Valency of the fusionprotein indicates the total number of binding arms or sites the fusionprotein has to antigen(s) or epitope(s); i.e., monovalent, bivalent,trivalent or multivalent. The multivalency of the antibody fusionprotein means that it can take advantage of multiple interactions inbinding to an antigen, thus increasing the avidity of binding to theantigen, or to different antigens. Specificity indicates how manydifferent types of antigen or epitope an antibody fusion protein is ableto bind; i.e., monospecific, bispecific, trispecific, multispecific.Using these definitions, a natural antibody, e.g., an IgG, is bivalentbecause it has two binding arms but is monospecific because it binds toone type of antigen or epitope. A monospecific, multivalent fusionprotein has more than one binding site for the same antigen or epitope.For example, a monospecific diabody is a fusion protein with two bindingsites reactive with the same antigen. The fusion protein may comprise amultivalent or multispecific combination of different antibodycomponents or multiple copies of the same antibody component. The fusionprotein may additionally comprise a therapeutic agent.

In some embodiments, the targeting moiety comprises a probody, such asthose disclosed in U.S. Pat. Nos. 8,518,404; 8,513,390; and US Pat.Appl. Pub. Nos.; 20120237977A1, 20120149061A1, 20130150558A1, thedisclosures of which are incorporated by reference in their entireties.

Probodies are monoclonal antibodies that are selectively activatedwithin the cancer microenvironment, focusing the activity of therapeuticantibodies to tumors and sparing healthy tissue.

In general, the probody comprises at least an antibody- or antibodyfragment thereof (collectively referred to as “AB”), capable ofspecifically binding a target, wherein the AB is modified by a maskingmoiety (MM). When the AB is modified with a MM and is in the presence ofthe target, specific binding of the AB to its target is reduced orinhibited, as compared to the specific binding of the AB not modifiedwith an MM or the specific binding of the parental AB to the target. Thedissociation constant (Kd) of the MM towards the AB is generally greaterthan the Kd of the AB towards the target. When the AB is modified with aMM and is in the presence of the target, specific binding of the AB toits target can be reduced or inhibited, as compared to the specificbinding of the AB not modified with an MM or the specific binding of theparental AB to the target. When an AB is coupled to or modified by a MM,the MM can ‘mask’ or reduce, or inhibit the specific binding of the ABto its target. When an AB is coupled to or modified by a MM, suchcoupling or modification can effect a structural change which reduces orinhibits the ability of the AB to specifically bind its target.

In some embodiments, the probody is an activatable antibodies (AAs)where the AB modified by an MM can further include one or more cleavablemoieties (CM). Such AAs exhibit activatable/switchable binding, to theAB's target. AAs generally include an antibody or antibody fragment(AB), modified by or coupled to a masking moiety (MM) and a modifiableor cleavable moiety (CM). In some embodiments, the CM contains an aminoacid sequence that serves as a substrate for a protease of interest. Inother embodiments, the CM provides a cysteine-cysteine disulfide bondthat is cleavable by reduction. In yet other embodiments the CM providesa photolytic substrate that is activatable by photolysis.

The CM and AB of the AA may be selected so that the AB represents abinding moiety for a target of interest, and the CM represents asubstrate for a protease that is co-localized with the target at atreatment site in a subject. Alternatively, or in addition, the CM is acysteine-cysteine disulfide bond that is cleavable as a result ofreduction of this disulfide bond. AAs contain at least one of aprotease-cleavable CM or a cysteine-cysteine disulfide bond, and in someembodiments, include both kinds of CMs. The AAs can alternatively orfurther include a photolabile substrate, activatable by a light source.The AAs disclosed herein find particular use where, for example, aprotease capable of cleaving a site in the CM is present at relativelyhigher levels in target-containing tissue of a treatment site (forexample diseased tissue; for example, for therapeutic treatment ordiagnostic treatment) than in tissue of non-treatment sites (for examplein healthy tissue). The AAs disclosed herein also find particular usewhere, for example, a reducing agent capable of reducing a site in theCM is present at relatively higher levels in target-containing tissue ofa treatment or diagnostic site than in tissue of non-treatmentnon-diagnostic sites. The AAs disclosed herein also find particular usewhere, for example, a light source, for example, by way of laser,capable of photolysing a site in the CM is introduced to atarget-containing tissue of a treatment or diagnostic site.

In some embodiments, AAs can provide for reduced toxicity and/or adverseside effects that could otherwise result from binding of the AB atnon-treatment sites if the AB were not masked or otherwise inhibitedfrom binding its target. Where the AA contains a CM that is cleavable bya reducing agent that facilitates reduction of a disulfide bond, the ABsof such AAs may be selected to exploit activation of an AB where atarget of interest is present at a desired treatment site characterizedby elevated levels of a reducing agent, such that the environment is ofa higher reduction potential than, for example, an environment of anon-treatment site.

In general, an AA can be designed by selecting an AB of interest andconstructing the remainder of the AA so that, when conformationallyconstrained, the MM provides for masking of the AB or reduction ofbinding of the AB to its target. Structural design criteria to be takeninto account to provide for this functional feature.

Anti-PD-1 Antibodies

In some embodiments, the TM is a monoclonal anti-PD-1 antibody.

Programmed Death-1 (“PD-1”) is a receptor of PD-L1 (also known as CD274,B7-H1, or B7-DC). PD-1 is an approximately 31 kD type I membrane proteinmember of the extended CD28/CTLA4 family of T cell regulators (Ishida,Y. et al. (1992) EMBO J. 11:3887-3895; US Pat. Appl. Pub. No.2007/0202100; 2008/0311117; 2009/00110667; U.S. Pat. Nos. 6,808,710;7,101,550; 7,488,802; 7,635,757; 7,722,868; PCT Publication No. WO01/14557). In comparison to CTLA4, PD-1 more broadly negativelyregulates immune responses.

PD-1 is expressed on activated T cells, B cells, and monocytes (Agata,Y. et al. (1996) Int. Immunol. 8(5):765-772; Yamazaki, T. et al. (2002J. Immunol. 169:5538-5545) and at low levels in natural killer (NK) Tcells (Nishimura, H. et al. (2000) J. Exp. Med. 191:891-898;Martin-Orozco, N. et al. (2007), Semin. Cancer Biol. 17(4):288-298).

The extracellular region of PD-1 consists of a single immunoglobulin(Ig) V domain with 23% identity to the equivalent domain in CTLA4(Martin-Orozco, N. et al. (2007) Semin. Cancer Biol. 17(4):288-298). Theextracellular IgV domain is followed by a transmembrane region and anintracellular tail. The intracellular tail contains two phosphorylationsites located in an immunoreceptor tyrosine-based inhibitory motif andan immunoreceptor tyrosine-based switch motif, which suggests that PD-1negatively regulates TCR signals (Ishida, Y. et al. (1992 EMBO J.11:3887-3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol. Immunother.56(5):739-745).

Antibodies capable of immunospecifically binding to murine PD-1 havebeen reported (see, e.g., Agata, T. et al. (1996) Int. Immunol.8(5):765-772).

Anti-PD-1 antibodies bind to PD-1 and enhance T-cell function toupregulate cell-mediated immune responses and for the treatment of Tcell dysfunctional disorders, such as tumor immunity.

In some embodiments, the anti-PD-1 antibody is MK-3475 (formerlyIambrolizumab, Merck), AMP-514, AMP-224 (MedImmune/AstraZeneca),BMS-936558 (MDX-1106, Bristol-Myers Squibb), or CT-011 (Curetech).

Pembrolizumab (MK-3475) is a humanized, monoclonal anti-PD-1 antibodydesigned to reactivate anti-tumor immunity. Pembrolizumab exerts dualligand blockade of the PD-1 pathway by inhibiting the interaction ofPD-1 on T cells with its ligands PD-L1 and PD-L2.

In some embodiments, the anti-PD-1 antibody is one of the antibodiesdisclosed in U.S. Pat. No. 8,354,509, and U.S. Pat. No. 8,168,757, thedisclosure of which is incorporated by reference in their entirety.

Nivolumab (also known as BMS-936558 or MDX1106, is a fully human IgG4monoclonal antibody developed by Bristol-Myers Squibb for the treatmentof cancer.

In some embodiments, the anti-PD-1 antibody is one of the antibodiesdisclosed in WO2004/056875, U.S. Pat. No. 7,488,802 and U.S. Pat. No.8,008,449, the disclosure of which is incorporated by reference in theirentirety.

AMP-514 and AMP-224 are an anti-programmed cell death 1 (PD-1)monoclonal antibody (mAb) developed by Amplimmune, which was acquired byMedImmune.

In some embodiments, the anti-PD-1 antibody is one of the antibodiesdisclosed in US Appl. Pub. No. 20140044738, the disclosure of which isincorporated by reference in their entirety.

In some embodiments, the six CDRs are: (A) the three light chain and thethree heavy chain CDRs of anti-PD-1 antibody 1E3; (B) the three lightchain and the three heavy chain CDRs of anti-PD-1 antibody 1E8; or (C)the three light chain and the three heavy chain CDRs of anti-PD-1antibody 1H3.

Pidilizumab (CT-011) is an anti-PD-1 monoclonal antibody developed byIsrael-based Curetech Ltd.

In some embodiments, the anti-PD-1 antibody is one of the antibodiesdisclosed in US Pat. Appl. Pub. Nos. 20080025980 and 20130022595, thedisclosure of which is incorporated by reference in their entirety.

Anti-PD-L1 Antibodies

In some embodiments, the TM is a monoclonal anti-PD-L1 antibody.

Programmed cell death 1 ligand 1 (PD-L1, also known as CD274 and B7-H1)is a ligand for PD-1, found on activated T cells, B cells, myeloid cellsand macrophages. Although there are two endogenous ligands for PD-1,PD-L1 and PD-L2, anti-tumor therapies have focused on anti-PD-L1antibodies. The complex of PD-1 and PD-L1 inhibits proliferation of CD8+T cells and reduces the immune response (Topalian et al., 2012, N Engl JMed 366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65).Anti-PD-L1 antibodies have been used for treatment of non-small celllung cancer, melanoma, colorectal cancer, renal-cell cancer, pancreaticcancer, gastric cancer, ovarian cancer, breast cancer, and hematologicmalignancies (Brahmer et al., N Eng J Med 366:2455-65; Ott et al., 2013,Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger etal., 2008, Clin Cancer Res 14:13044-51). PD-L1 is a B7 family memberthat is expressed on many cell types, including APCs and activated Tcells (Yamazaki et al. (2002) J. Immunol. 169:5538). PD-L1 binds to bothPD-1 and B7-1. Both binding of T-cell-expressed B7-1 by PD-L1 andbinding of T-cell-expressed PD-L1 by B7-1 result in T cell inhibition(Butte et al. (2007) Immunity 27:111). There is also evidence that, likeother B7 family members, PD-L1 can also provide costimulatory signals toT cells (Subudhi et al. (2004) J. Clin. Invest. 113:694; Tamura et al.(2001) Blood 97:1809).

By “PD-L1” herein is meant to include any variants or isoforms which arenaturally expressed by cells, and/or fragments thereof having at leastone biological activity of the full-length polypeptide, unless otherwiseexpressly defined. In addition, the term “PD-L1” includes PD-L1 (Freemanet al. (2000) J. Exp. Med. 192:1027) and any variants or isoforms whichare naturally expressed by cells, and/or fragments thereof having atleast one biological activity of the full-length polypeptides. Forexample, PD-L1 sequences from different species, including humans, arewell known in the art (see, for example, herein incorporated in theirentirety by reference, Chen et al., U.S. Pat. No. 6,803,192, whichdiscloses human and mouse PD-L1 sequences; Wood et al., U.S. Pat. No.7,105,328, which discloses human PD-L1 sequences.

Anti-PD-L1 antibodies bind to PD-L1 and enhance T-cell function toupregulate cell-mediated immune responses and for the treatment of Tcell dysfunctional disorders, such as tumor immunity.

In some embodiments, the anti-PD-L1 antibody is MPDL3280A andYW243.55.S70, (Genentech/Roche), MEDI-4736 (MedImmune/AstraZeneca),BMS-936559 (MDX-1105, Bristol-Myers Squibb), and MSB0010718C (EMDSerono/Merck KGaA).

MPDL3280A (Genentech) is an engineered anti-PD-L1 antibody designed totarget PD-L1 expressed on tumor cells and tumor-infiltrating immunecells. MPDL3280A is designed to prevent PD-L1 from binding to PD-1 andB7.1. This blockade of PD-L1 may enable the activation of T cells,restoring their ability to detect and attack tumor cells. MPDL3280Acontains an engineered fragment crystallizable (Fc) domain designed tooptimize efficacy and safety by minimizing antibody-dependent cellularcytotoxicity (ADCC).

In some embodiments, the anti-PD-L1 antibody is one of the antibodiesdisclosed in U.S. Pat. No. 7,943,743, the disclosure of which isincorporated by reference in their entirety.

BMS-936559 (MDX-1105, Bristol-Myers Squibb) is a fully human IgG4anti-PD-L1 mAb that inhibits the binding of the PD-L1 ligand to bothPD-1 and CD80.

In some embodiments, the anti-PD-L1 antibody is one of the antibodiesdisclosed in U.S. Pat. No. 7,943,743, the disclosure of which isincorporated by reference in their entirety.

MSB0010718C (EMD Serono of Merck KGaA) is fully human IgG1 monoclonalantibody that binds to PD-L1.

In some embodiments, the anti-PD-L1 antibody is one of the antibodiesdisclosed in WO 2013079174 A1, the disclosure of which is incorporatedby reference in their entirety.

MEDI4736 (MedImmune/AstraZeneca) is a human IgG1 antibody which bindsspecifically to PD-L1, preventing binding to PD-1 and CD80.

In some embodiments, the anti-PD-L1 antibody is one of the antibodiesdisclosed in WO 2011066389 A1 and U.S. Pat. No. 8,779,108, thedisclosure of which is incorporated by reference in their entirety.

In some embodiments, the anti-PD-L1 antibody is one of the antibodiesdisclosed in U.S. Pat. No. 8,552,154, the disclosure of which isincorporated by reference in their entirety.

In some embodiments, the targeting moiety comprises a Fab, Fab′,F(ab′)2, single domain antibody, T and Abs dimer, Fv, scFv, dsFv,ds-scFv, Fd, linear antibody, minibody, diabody, bispecific antibodyfragment, bibody, tribody, sc-diabody, kappa (lamda) body, BiTE, DVD-Ig,SIP, SMIP, DART, or an antibody analogue comprising one or more CDRs.

PD-L/PD-1 Axis Antagonist Comprising a Targeting Moiety

In some aspects, the PD-L/PD-1 Axis antagonist is a targeted therapeuticcomprise a targeting moiety, such as an ADC.

By “targeting moiety (TM)” or “targeting agent” here in is meant amolecule, complex, or aggregate, that binds specifically or selectivelyto a target molecule, cell, particle, tissue or aggregate, whichgenerally is referred to as a “target” or a “marker,” and these arediscussed in further detail herein.

In some embodiments, the targeting moiety comprises an immunoglobulin, aprotein, a peptide, a small molecule, a nanoparticle, or a nucleic acid.

Exemplary targeting agents such as antibodies (e.g., chimeric, humanizedand human), ligands for receptors, lecitins, and saccharides, andsubstrate for certain enzymes are recognized in the art and are usefulwithout limitation in practicing the present invention. Other targetingagents include a class of compounds that do not include specificmolecular recognition motifs include nanoparticles, macromolecules suchas poly(ethylene glycol), polysaccharide, and polyamino acids which addmolecular mass to the activating moiety. The additional molecular massaffects the pharmacokinetics of the activating moiety, e.g., serumhalf-life.

In some embodiments, a targeting moiety is an antibody, antibodyfragment, bispecific antibody or other antibody-based molecule orcompound. However, other examples of targeting moieties are known in theart and may be used, such as aptamers, avimers, receptor-bindingligands, nucleic acids, biotin-avidin binding pairs, binding peptides orproteins, etc. The terms “targeting moiety” and “binding moiety” areused synonymously herein.

By “target” or “marker” herein is meant any entity that is capable ofspecifically binding to a particular targeting moiety. In someembodiments, targets are specifically associated with one or moreparticular cell or tissue types. In some embodiments, targets arespecifically associated with one or more particular disease states. Insome embodiments, targets are specifically associated with one or moreparticular developmental stages. For example, a cell type specificmarker is typically expressed at levels at least 2 fold greater in thatcell type than in a reference population of cells. In some embodiments,the cell type specific marker is present at levels at least 3 fold, atleast 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, atleast 100 fold, or at least 1,000 fold greater than its averageexpression in a reference population. Detection or measurement of a celltype specific marker may make it possible to distinguish the cell typeor types of interest from cells of many, most, or all other types. Insome embodiments, a target can comprise a protein, a carbohydrate, alipid, and/or a nucleic acid, as described herein.

A substance is considered to be “targeted” for the purposes describedherein if it specifically binds to a nucleic acid targeting moiety. Insome embodiments, a nucleic acid targeting moiety specifically binds toa target under stringent conditions. An inventive complex or compoundcomprising targeting moiety is considered to be “targeted” if thetargeting moiety specifically binds to a target, thereby delivering theentire complex or compound composition to a specific organ, tissue,cell, extracellular matrix component, and/or intracellular compartment.

In certain embodiments, compound in accordance with the presentinvention comprise a targeting moiety which specifically binds to one ormore targets (e.g. antigens) associated with an organ, tissue, cell,extracellular matrix component, and/or intracellular compartment. Insome embodiments, compounds comprise a targeting moiety whichspecifically binds to targets associated with a particular organ ororgan system. In some embodiments, compounds in accordance with thepresent invention comprise a nuclei targeting moiety which specificallybinds to one or more intracellular targets (e.g. organelle,intracellular protein). In some embodiments, compounds comprise atargeting moiety which specifically binds to targets associated withdiseased organs, tissues, cells, extracellular matrix components, and/orintracellular compartments. In some embodiments, compounds comprise atargeting moiety which specifically binds to targets associated withparticular cell types (e.g. endothelial cells, cancer cells, malignantcells, prostate cancer cells, etc.).

In some embodiments, compounds in accordance with the present inventioncomprise a targeting moiety which binds to a target that is specific forone or more particular tissue types (e.g. liver tissue vs. prostatetissue). In some embodiments, compounds in accordance with the presentinvention comprise a targeting moiety which binds to a target that isspecific for one or more particular cell types (e.g. T cells vs. Bcells). In some embodiments, compounds in accordance with the presentinvention comprise a targeting moiety which binds to a target that isspecific for one or more particular disease states (e.g. tumor cells vs.healthy cells). In some embodiments, compounds in accordance with thepresent invention comprise a targeting moiety which binds to a targetthat is specific for one or more particular developmental stages (e.g.stem cells vs. differentiated cells).

In some embodiments, a target may be a marker that is exclusively orprimarily associated with one or a few cell types, with one or a fewdiseases, and/or with one or a few developmental stages. A cell typespecific marker is typically expressed at levels at least 2 fold greaterin that cell type than in a reference population of cells which mayconsist, for example, of a mixture containing cells from a plurality(e.g., 5-10 or more) of different tissues or organs in approximatelyequal amounts. In some embodiments, the cell type specific marker ispresent at levels at least 3 fold, at least 4 fold, at least 5 fold, atleast 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, atleast 10 fold, at least 50 fold, at least 100 fold, or at least 1000fold greater than its average expression in a reference population.Detection or measurement of a cell type specific marker may make itpossible to distinguish the cell type or types of interest from cells ofmany, most, or all other types.

In some embodiments, a target comprises a protein, a carbohydrate, alipid, and/or a nucleic acid. In some embodiments, a target comprises aprotein and/or characteristic portion thereof, such as a tumor-marker,integrin, cell surface receptor, transmembrane protein, intercellularprotein, ion channel, membrane transporter protein, enzyme, antibody,chimeric protein, glycoprotein, etc. In some embodiments, a targetcomprises a carbohydrate and/or characteristic portion thereof, such asa glycoprotein, sugar (e.g., monosaccharide, disaccharide,polysaccharide), glycocalyx (i.e., the carbohydrate-rich peripheral zoneon the outside surface of most eukaryotic cells) etc. In someembodiments, a target comprises a lipid and/or characteristic portionthereof, such as an oil, fatty acid, glyceride, hormone, steroid (e.g.,cholesterol, bile acid), vitamin (e.g. vitamin E), phospholipid,sphingolipid, lipoprotein, etc. In some embodiments, a target comprisesa nucleic acid and/or characteristic portion thereof, such as a DNAnucleic acid; RNA nucleic acid; modified DNA nucleic acid; modified RNAnucleic acid; nucleic acid that includes any combination of DNA, RNA,modified DNA, and modified RNA.

Numerous markers are known in the art. Typical markers include cellsurface proteins, e.g., receptors. Exemplary receptors include, but arenot limited to, the transferrin receptor; LDL receptor; growth factorreceptors such as epidermal growth factor receptor family members (e.g.,EGFR, Her2, Her3, Her4) or vascular endothelial growth factor receptors,cytokine receptors, cell adhesion molecules, integrins, selectins, andCD molecules. The marker can be a molecule that is present exclusivelyor in higher amounts on a malignant cell, e.g., a tumor antigen.

In some embodiments, the targeting moiety binds to a tumor cellspecifically or preferably in comparison to a non-tumor cell.

The binding of target moiety to tumor cell can be measured using assaysknown in the art.

In some embodiments, the tumor cell is of a carcinoma, a sarcoma, alymphoma, a myeloma, or a central nervous system cancer.

In some embodiments, the targeting moiety is capable of binding to atumor antigen specifically or preferably in comparison to a non-tumorantigen.

In certain specific embodiments, a target is a tumor marker. In someembodiments, a tumor marker is an antigen that is present in a tumorthat is not present in normal organs, tissues, and/or cells. In someembodiments, a tumor marker is an antigen that is more prevalent in atumor than in normal organs, tissues, and/or cells. In some embodiments,a tumor marker is an antigen that is more prevalent in malignant cancercells than in normal cells.

In some embodiments, the targeting moiety comprises folic acid or aderivative thereof.

In recent years, research on folic acid had made great progress. Folicacid is a small molecule vitamin that is necessary for cell division.Tumor cells divide abnormally and there is a high expression of folatereceptor (FR) on tumor cell surface to capture enough folic acid tosupport cell division.

Data indicate FR expression in tumor cells is 20-200 times higher thannormal cells. The expression rate of FR in various malignant tumors are:82% in ovarian cancer, 66% in non-small cell lung cancer, 64% in kidneycancer, 34% in colon cancer, and 29% in breast cancer (Xia W, Low P S.Late-targeted therapies for cancer. J Med Chem. 2010; 14; 53(19):6811-24). The expression rate of FA and the degree of malignancy ofepithelial tumor invasion and metastasis is positively correlated. FAenters cell through FR mediated endocytosis, and FA through its carboxylgroup forms FA complexes with drugs which enter the cells. Under acidicconditions (pH value of 5), FR separates from the FA, and FA releasesdrugs into the cytoplasm.

Clinically, the system can be used to deliver drugs selectively attackthe tumor cells. Folic acid has small molecular weight, hasnon-immunogenicity and high stability, and is inexpensive to synthesis.More importantly, chemical coupling between the drug and the carrier issimple, and as such using FA as targeting molecule to construct drugdelivery system has become a research hotspot for cancer treatment.Currently EC 145 (FA chemotherapy drug conjugate compound) that is inclinical trials can effectively attack cancer cells (Pribble P andEdelman M J. EC 145: a novel targeted agent for adenocarcinoma of thelung. Expert Opin. Investig. Drugs (2012) 21:755-761).

In some embodiments, the targeting moiety comprises extracellulardomains (ECD) or soluble form of PD-1, PDL-1, CTLA4, CD47, BTLA, KIR,TIM3, 4-1BB, and LAG3, full length of partial of a surface ligandAmphiregulin, Betacellulin, EGF, Ephrin, Epigen, Epiregulin, IGF,Neuregulin, TGF, TRAIL, or VEGF.

In some embodiments, the targeting moiety comprises a Fab, Fab′,F(ab′)2, single domain antibody, T and Abs dimer, Fv, scFv, dsFv,ds-scFv, Fd, linear antibody, minibody, diabody, bispecific antibodyfragment, bibody, tribody, sc-diabody, kappa (lamda) body, BiTE, DVD-Ig,SIP, SMIP, DART, or an antibody analogue comprising one or more CDRs.

In some embodiments, the targeting moiety is an antibody, or antibodyfragment, that is selected based on its specificity for an antigenexpressed on a target cell, or at a target site, of interest. A widevariety of tumor-specific or other disease-specific antigens have beenidentified and antibodies to those antigens have been used or proposedfor use in the treatment of such tumors or other diseases. Theantibodies that are known in the art can be used in the compounds of theinvention, in particular for the treatment of the disease with which thetarget antigen is associated. Examples of target antigens (and theirassociated diseases) to which an antibody-linker-drug conjugate of theinvention can be targeted include: CD2, CD19, CD20, CD22, CD27, CD33,CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-1BB, 5T4,AGS-5, AGS-16, Angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3,BT-062, BTLA, CAIX, Carcinoembryonic antigen, CTLA4, Cripto, ED-B,ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP,Fibronectin, Folate Receptor, Ganglioside GM3, GD2,glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100,gpA33, GPNMB, ICOS, IGF1R, Integrin αν, Integrin ανβ, KIR, LAG-3, LewisY, Mesothelin, c-MET, MN Carbonic anhydrase IX, MUC1, MUC16, Nectin-4,NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2,SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILR1, TRAILR2,VEGFR-1, VEGFR-2, VEGFR-3.

In some embodiments, the targeting moiety comprises a particle (targetparticle), preferably a nanoparticle, optionally a targeted nanoparticlethat attached to a targeting molecule that can binds specifically orpreferably to a target. In some embodiments, the targeting particle byitself guides die compound of the present invention (such as byenrichment in tumor cells or tissue) and there is no additionaltargeting molecules attached therein.

By “nanoparticle” herein is meant any particle having a diameter of lessthan 1000 nm. In some embodiments, a therapeutic agent and/or targetingmolecule can be associated with the polymeric matrix. In someembodiments, the targeting molecule can be covalently associated withthe surface of a polymeric matrix. In some embodiments, covalentassociation is mediated by a linker. In some embodiments, thetherapeutic agent can be associated with the surface of, encapsulatedwithin, surrounded by, and/or dispersed throughout the polymeric matrix.U.S. Pat. No. 8,246,968, which is incorporated in its entirety.

In general, nanoparticles of the present invention comprise any type ofparticle. Any particle can be used in accordance with the presentinvention. In some embodiments, particles are biodegradable andbiocompatible. In general, a biocompatible substance is not toxic tocells. In some embodiments, a substance is considered to bebiocompatible if its addition to cells results in less than a certainthreshold of cell death. In some embodiments, a substance is consideredto be biocompatible if its addition to cells does not induce adverseeffects. In general, a biodegradable substance is one that undergoesbreakdown under physiological conditions over the course of atherapeutically relevant time period (e.g., weeks, months, or years). Insome embodiments, a biodegradable substance is a substance that can bebroken down by cellular machinery. In some embodiments, a biodegradablesubstance is a substance that can be broken down by chemical processes.In some embodiments, a particle is a substance that is bothbiocompatible and biodegradable. In some embodiments, a particle is asubstance that is biocompatible, but not biodegradable. In someembodiments, a particle is a substance that is biodegradable, but notbiocompatible.

In some embodiments, particles are greater in size than the renalexcretion limit (e.g. particles having diameters of greater than 6 nm).In some embodiments, particles are small enough to avoid clearance ofparticles from the bloodstream by the liver (e.g. particles havingdiameters of less than 1000 nm). In general, phytochemical features ofparticles should allow a targeted particle to circulate longer in plasmaby decreasing renal excretion and liver clearance.

It is often desirable to use a population of particles that isrelatively uniform in terms of size, shape, and/or composition so thateach particle has similar properties. For example, at least 80%, atleast 90%, or at least 95% of the particles may have a diameter orgreatest dimension that falls within 5%, 10%, or 20% of the averagediameter or greatest dimension. In some embodiments, a population ofparticles may be heterogeneous with respect to size, shape, and/orcomposition.

Zeta potential is a measurement of surface potential of a particle. Insome embodiments, particles have a zeta potential ranging between −50 mVand +50 mV. In some embodiments, particles have a zeta potential rangingbetween −25 mV and +25 mV. In some embodiments, particles have a zetapotential ranging between −10 mV and +10 mV. In some embodiments,particles have a zeta potential ranging between −5 mV and +5 mV. In someembodiments, particles have a zeta potential ranging between 0 mV and+50 mV. In some embodiments, particles have a zeta potential rangingbetween 0 mV and +25 mV.

In some embodiments, particles have a zeta potential ranging between 0mV and +10 mV. In some embodiments, particles have a zeta potentialranging between 0 mV and +5 mV. In some embodiments, particles have azeta potential ranging between −50 mV and 0 mV. In some embodiments,particles have a zeta potential ranging between −25 mV and 0 mV. In someembodiments, particles have a zeta potential ranging between −10 mV and0 mV. In some embodiments, particles have a zeta potential rangingbetween −5 mV and 0 mV. In some embodiments, particles have asubstantially neutral zeta potential (i.e. approximately 0 mV).

A variety of different particles can be used in accordance with thepresent invention. In some embodiments, particles are spheres orspheroids. In some embodiments, particles are spheres or spheroids. Insome embodiments, particles are flat or plate-shaped. In someembodiments, particles are cubes or cuboids. In some embodiments,particles are ovals or ellipses. In some embodiments, particles arecylinders, cones, or pyramids.

In some embodiments, particles are microparticles (e.g. microspheres).In general, a “microparticle” refers to any particle having a diameterof less than 1000 μm. In some embodiments, particles are picoparticles(e.g. picospheres). In general, a “picoparticle” refers to any particlehaving a diameter of less than 1 nm. In some embodiments, particles areliposomes. In some embodiments, particles are micelles.

Particles can be solid or hollow and can comprise one or more layers(e.g., nanoshells, nanorings). In some embodiments, each layer has aunique composition and unique properties relative to the other layers).For example, particles may have a core/shell structure, wherein the coreis one layer and the shell is a second layer. Particles may comprise aplurality of different layers. In some embodiments, one layer may besubstantially cross-linked, a second layer is not substantiallycross-linked, and so forth. In some embodiments, one, a few, or all ofthe different layers may comprise one or more therapeutic or diagnosticagents to be delivered. In some embodiments, one layer comprises anagent to be delivered, a second layer does not comprise an agent to bedelivered, and so forth. In some embodiments, each individual layercomprises a different agent or set of agents to be delivered.

In some embodiments, a particle is porous, by which is meant that theparticle contains holes or channels, which are typically small comparedwith the size of a particle. For example, a particle may be a poroussilica particle, e.g., a mesoporous silica nanoparticle or may have acoating of mesoporous silica (Lin et al., 2005, J. Am. Chem. Soc.,17:4570). Particles may have pores ranging from about 1 nm to about 50nm in diameter, e.g., between about 1 and 20 nm in diameter. Betweenabout 10% and 95% of the volume of a particle may consist of voidswithin the pores or channels.

Particles may have a coating layer. Use of a biocompatible coating layercan be advantageous, e.g., if the particles contain materials that aretoxic to cells. Suitable coating materials include, but are not limitedto, natural proteins such as bovine serum albumin (BSA), biocompatiblehydrophilic polymers such as polyethylene glycol (PEG) or a PEGderivative, phospholipid-(PEG), silica, lipids, polymers, carbohydratessuch as dextran, other nanoparticles that can be associated withinventive nanoparticles etc.

Coatings may be applied or assembled in a variety of ways such as bydipping, using a layer-by-layer technique, by self-assembly,conjugation, etc. Self-assembly refers to a process of spontaneousassembly of a higher order structure that relies on the naturalattraction of the components of the higher order structure (e.g.,molecules) for each other. It typically occurs through random movementsof the molecules and formation of bonds based on size, shape,composition, or chemical properties.

Examples of polymers include polyalkylenes (e.g. polyethylenes),polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhydrides (e.g.poly(sebacic anhydride)), polyhydroxyacids (e.g.poly(β-hydroxyalkanoate)), polyfumarates, polycaprolactones, polyamides(e.g. polycaprolactam), polyacetals, polyethers, polyesters (e.g.polylactide, polyglycolide), poly(orthoesters), polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, and polyamines. In someembodiments, polymers in accordance with the present invention includepolymers which have been approved for use in humans by the U.S. Food andDrug Administration (FDA) under 21 C.F.R. § 177.2600, including but notlimited to polyesters (e.g. polylactic acid, polyglycolic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g. poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, particles can be non-polymeric particles (e.g.metal particles, quantum dots, ceramic particles, polymers comprisinginorganic materials, bone-derived materials, bone substitutes, viralparticles, etc.). In some embodiments, a therapeutic or diagnostic agentto be delivered can be associated with the surface of such anon-polymeric particle. In some embodiments, a non-polymeric particle isan aggregate of non-polymeric components, such as an aggregate of metalatoms (e.g. gold atoms). In some embodiments, a therapeutic ordiagnostic agent to be delivered can be associated with the surface ofand/or encapsulated within, surrounded by, and/or dispersed throughoutan aggregate of non-polymeric components.

Particles (e.g. nanoparticles, microparticles) may be prepared using anymethod known in the art. For example, particulate formulations can beformed by methods as nanoprecipitation, flow focusing fluidic channels,spray drying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanoparticles have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843).

Methods for making microparticles for delivery of encapsulated agentsare described in the literature (see, e.g., Doubrow, Ed., “Microcapsulesand Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton,1992; Mathiowitz et al., 1987, J. Control. Release, 5:13; Mathiowitz etal., 1987, Reactive Polymers, δ: 275; and Mathiowitz et al., 1988, J.Appl. Polymer Sci., 35:755).

In some embodiments, the targeting moiety comprises an nucleic acidtargeting moiety.

In general, a nucleic acid targeting moiety is any polynucleotide thatbinds to a component associated with an organ, tissue, cell,extracellular matrix component, and/or intracellular compartment (thetarget).

In some embodiments, the nucleic acid targeting moieties are aptamers.

An aptamer is typically a polynucleotide that binds to a specific targetstructure that is associated with a particular organ, tissue, cell,extracellular matrix component, and/or intracellular compartment. Ingeneral, the targeting function of the aptamer is based on thethree-dimensional structure of the aptamer. In some embodiments, bindingof an aptamer to a target is typically mediated by the interactionbetween the two- and/or three-dimensional structures of both the aptamerand the target. In some embodiments, binding of an aptamer to a targetis not solely based on the primary sequence of the aptamer, but dependson the three-dimensional structure(s) of the aptamer and/or target. Insome embodiments, aptamers bind to their targets via complementaryWatson-Crick base pairing which is interrupted by structures (e.g.hairpin loops) that disrupt base pairing.

In some embodiments, the nucleic acid targeting moieties are spiegelmers(PCT Publications WO 98/08856, WO 02/100442, and WO 06/117217). Ingeneral, spiegelmers are synthetic, mirror-image nucleic acids that canspecifically bind to a target (i.e. mirror image aptamers). Spiegelmersare characterized by structural features which make them not susceptibleto exo- and endo-nucleases.

One of ordinary skill in the art will recognize that any nucleic acidtargeting moiety (e.g. aptamer or spiegelmer) that is capable ofspecifically binding to a target can be used in accordance with thepresent invention. In some embodiments, nucleic acid targeting moietiesto be used in accordance with the present invention may target a markerassociated with a disease, disorder, and/or condition. In someembodiments, nucleic acid targeting moieties to be used in accordancewith the present invention may target cancer-associated targets. In someembodiments, nucleic acid targeting moieties to be used in accordancewith the present invention may target tumor markers. Any type of cancerand/or any tumor marker may be targeted using nucleic acid targetingmoieties in accordance with the present invention. To give but a fewexamples, nucleic acid targeting moieties may target markers associatedwith prostate cancer, lung cancer, breast cancer, colorectal cancer,bladder cancer, pancreatic cancer, endometrial cancer, ovarian cancer,bone cancer, esophageal cancer, liver cancer, stomach cancer, braintumors, cutaneous melanoma, and/or leukemia.

Nucleic acids of the present invention (including nucleic acid nucleicacid targeting moieties and/or functional RNAs to be delivered, e.g.,RNAi-inducing entities, ribozymes, tRNAs, etc., described in furtherdetail below) may be prepared according to any available techniqueincluding, but not limited to chemical synthesis, enzymatic synthesis,enzymatic or chemical cleavage of a longer precursor, etc. Methods ofsynthesizing RNAs are known in the art (see, e.g., Gait, M. J. (ed.)Oligonucleotide synthesis: a practical approach. Oxford [Oxfordshire],Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.)Oligonucleotide synthesis: methods and applications, Methods inmolecular biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press,2005).

The nucleic acid that forms the nucleic acid nucleic acid targetingmoiety may comprise naturally occurring nucleosides, modifiednucleosides, naturally occurring nucleosides with hydrocarbon linkers(e.g., an alkylene) or a polyether linker (e.g., a PEG linker) insertedbetween one or more nucleosides, modified nucleosides with hydrocarbonor PEG linkers inserted between one or more nucleosides, or acombination of thereof. In some embodiments, nucleotides or modifiednucleotides of the nucleic acid nucleic acid targeting moiety can bereplaced with a hydrocarbon linker or a poly ether linker provided thatthe binding affinity and selectivity of the nucleic acid nucleic acidtargeting moiety is not substantially reduced by the substitution (e.g.,the dissociation constant of the nucleic acid nucleic acid targetingmoiety for the target should not be greater than about 1×10⁻³ M).

It will be appreciated by those of ordinary skill in the art thatnucleic acids in accordance with the present invention may comprisenucleotides entirely of the types found in naturally occurring nucleicacids, or may instead include one or more nucleotide analogs or have astructure that otherwise differs from that of a naturally occurringnucleic acid. U.S. Pat. Nos. 6,403,779; 6,399,754; 6,225,460; 6,127,533;6,031,086; 6,005,087; 5,977,089; and references therein disclose a widevariety of specific nucleotide analogs and modifications that may beused. See Crooke, S. (ed.) Antisense Drug Technology: Principles,Strategies, and Applications (1st ed), Marcel Dekker; ISBN: 0824705661;1st edition (2001) and references therein. For example, 2′-modificationsinclude halo, alkoxy and allyloxy groups. In some embodiments, the 2′-OHgroup is replaced by a group selected from H, OR, R, halo, SH, SR, NH2,NHR, NR2 or CN, wherein R is C1-C6 alkyl, alkenyl, or alkynyl, and halois F, Cl, Br, or I. Examples of modified linkages includephosphorothioate and 5′-N-phosphoramidite linkages.

Nucleic acids comprising a variety of different nucleotide analogs,modified backbones, or non-naturally occurring internucleoside linkagescan be utilized in accordance with the present invention.

Nucleic acids of the present invention may include natural nucleosides(i.e., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) ormodified nucleosides. Examples of modified nucleotides include basemodified nucleoside (e.g., aracytidine, inosine, isoguanosine,nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine,2-thiothymidine, 3-deaza-5-azacytidine, 2′-deoxyuridine, 3-nitorpyrrole,4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine,2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine,6-azauridine, 6-chloropurine, 7-deazaadenosine, 7-deazaguanosine,8-azaadenosine, 8-azidoadenosine, benzimidazole, M1-methyladenosine,pyrrolo-pyrimidino, 2-amino-6-chloropurine, 3-methyl adenosine,5-propynylcytidine, 5-propynyluridine, 5-bromouridine, 5-fluorouridine,5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically orbiologically modified bases (e.g., methylated bases), modified sugars(e.g., 2′-fluororibose, 2′-aminoribose, 2′-azidoribose,2′-O-methylribose, L-enantiomeric nucleosides arabinose, and hexose),modified phosphate groups (e.g., phosphorothioates and5′-N-phosphoramidite linkages), and combinations thereof. Natural andmodified nucleotide monomers for the chemical synthesis of nucleic acidsare readily available. In some cases, nucleic acids comprising suchmodifications display improved properties relative to nucleic acidsconsisting only of naturally occurring nucleotides. In some embodiments,nucleic acid modifications described herein are utilized to reduceand/or prevent digestion by nucleases (e.g. exonucleases, endonucleases,etc.). For example, the structure of a nucleic acid may be stabilized byincluding nucleotide analogs at the 3′ end of one or both strands orderto reduce digestion.

Modified nucleic acids need not be uniformly modified along the entirelength of the molecule. Different nucleotide modifications and/orbackbone structures may exist at various positions in the nucleic acid.One of ordinary skill in the art will appreciate that the nucleotideanalogs or other modification(s) may be located at any position(s) of anucleic acid such that the function of the nucleic acid is notsubstantially affected. To give but one example, modifications may belocated at any position of a nucleic acid targeting moiety such that theability of the nucleic acid targeting moiety to specifically bind to thetarget is not substantially affected. The modified region may be at the5′-end and/or the 3′-end of one or both strands. For example, modifiednucleic acid targeting moieties in which approximately 1-5 residues atthe 5′ and/or 3′ end of either of both strands are nucleotide analogsand/or have a backbone modification have been employed. The modificationmay be a 5′ or 3′ terminal modification. One or both nucleic acidstrands may comprise at least 50% unmodified nucleotides, at least 80%unmodified nucleotides, at least 90% unmodified nucleotides, or 100%unmodified nucleotides.

Nucleic acids in accordance with the present invention may, for example,comprise a modification to a sugar, nucleoside, or internucleosidelinkage such as those described in U.S. Patent Application Publications2003/0175950, 2004/0192626, 2004/0092470, 2005/0020525, and2005/0032733. The present invention encompasses the use of any nucleicacid having any one or more of the modification described therein. Forexample, a number of terminal conjugates, e.g., lipids such ascholesterol, lithocholic acid, aluric acid, or long alkyl branchedchains have been reported to improve cellular uptake. Analogs andmodifications may be tested using, e.g., using any appropriate assayknown in the art, for example, to select diose that result in improveddelivery of a therapeutic or diagnostic agent, improved specific bindingof an nucleic acid targeting moiety to a target, etc. In someembodiments, nucleic acids in accordance with the present invention maycomprise one or more non-natural nucleoside linkages. In someembodiments, one or more internal nucleotides at the 3′-end, 5′-end, orboth 3′- and 5′-ends of the nucleic acid targeting moiety are invertedto yield a linkage such as a 3′-3′ linkage or a 5′-5′ linkage.

In some embodiments, nucleic acids in accordance with the presentinvention are not synthetic, but are naturally-occurring entities thathave been isolated from their natural environments.

Any method can be used to design novel nucleic acid targeting moieties(see, e.g., U.S. Pat. Nos. 6,716,583; 6,465,189; 6,482,594; 6,458,543;6,458,539; 6,376,190; 6,344,318; 6,242,246; 6,184,364; 6,001,577;5,958,691; 5,874,218; 5,853,984; 5,843,732; 5,843,653; 5,817,785;5,789,163; 5,763,177; 5,696,249; 5,660,985; 5,595,877; 5,567,588; and5,270,163; and U.S. Patent Application Publications 2005/0069910,2004/0072234, 2004/0043923, 2003/0087301, 2003/0054360, and2002/0064780). The present invention provides methods for designingnovel nucleic acid targeting moieties. The present invention furtherprovides methods for isolating or identifying novel nucleic acidtargeting moieties from a mixture of candidate nucleic acid targetingmoieties.

Nucleic acid targeting moieties that bind to a protein, a carbohydrate,a lipid, and/or a nucleic acid can be designed and/or identified. Insome embodiments, nucleic acid targeting moieties can be designed and/oridentified for use in the complexes of the invention that bind toproteins and/or characteristic portions thereof, such as tumor-markers,integrins, cell surface receptors, transmembrane proteins, intercellularproteins, ion channels, membrane transporter proteins, enzymes,antibodies, chimeric proteins etc. In some embodiments, nucleic acidtargeting moieties can be designed and/or identified for use in thecomplexes of the invention that bind to carbohydrates and/orcharacteristic portions thereof, such as glycoproteins, sugars (e.g.,monosaccharides, disaccharides and polysaccharides), glycocalyx (i.e.,the carbohydrate-rich peripheral zone on the outside surface of mosteukaryotic cells) etc. In some embodiments, nucleic acid targetingmoieties can be designed and/or identified for use in the complexes ofthe invention that bind to lipids and/or characteristic portionsthereof, such as oils, saturated fatty acids, unsaturated fatty acids,glycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins(e.g. vitamin E), phospholipids, sphingolipids, lipoproteins etc. Insome embodiments, nucleic acid targeting moieties can be designed and/oridentified for use in the complexes of the invention that bind tonucleic acids and/or characteristic portions thereof, such as DNAnucleic acids; RNA nucleic acids; modified DNA nucleic acids; modifiedRNA nucleic acids; and nucleic acids that include any combination ofDNA, RNA, modified DNA, and modified RNA; etc.

Nucleic acid targeting moieties (e.g. aptamers or spiegelmers) may bedesigned and/or identified using any available method. In someembodiments, nucleic acid targeting moieties are designed and/oridentified by identifying nucleic acid targeting moieties from acandidate mixture of nucleic acids. Systemic Evolution of Ligands byExponential Enrichment (SELEX), or a variation thereof, is a commonlyused method of identifying nucleic acid targeting moieties that bind toa target from a candidate mixture of nucleic acids.

Nucleic acid targeting moieties that bind selectively to any target canbe isolated by the SELEX process, or a variation thereof, provided thatthe target can be used as a target in the SELEX process.

B. Immunotherapeutics

In general, the combination or composition of the present inventioncomprises an immunotherapeutic.

By “immunotherapeutics” herein is meant a compound, a molecule, or anagent that is capable of stimulating or enhancing the body's immunesystem or tumor cells. Immunotherapeutics are used for the treatment ofdisease by inducing, enhancing, or suppressing an immune response.Immunotherapeutics of the present invention generally are designed toelicit or amplify an immune response, rather than suppress an immuneresponse.

In general, the immunotherapeutics of the present invention act,directly or indirectly, on toll like receptors,nucleotide-oligomerization domain-like receptors, RIG-I-Like receptors,c-type lectin receptors, or cytosolic DNA Sensors, or a combinationthereof. Particually, the immunotherapeutics of the present inventionare capable of activating a human plasmacytoid dendritic cell, myeloiddendritic cell, NK cell, or tumor cell, or a combination thereof.

In some embodiments, the immunotherapeutics of the present inventionactivate human immune cells, including but not limited to dendriticcells, macrophages, monocytes, myeloid-derived suppressor cells, NKcells, B cells, T cells, or tumor cells, or a combination thereof.

Dendritic cells are the most powerful antigen-presenting cells.Dendritic cells play an essential role for the initiation of both innateand adaptive immune responses. Dendritic cells also play a key role inthe induction and maintenance of immune tolerance.

By “dendritic cells” (DC) herein is meant a heterogeneous cellpopulation including two main subtypes: namely, myeloid DC (mDC) andplasmacytoid DC (pDC) (Steinman et al., 1979, J. Exp. Med., 149, 1-16).These two blood DC subsets were originally differentiated by theirexpression of CD11c (integrin complement receptor) and CD123 (IL-3Rα).Each of the pDC and mDC populations constitutes between about 0.2 toabout 0.6% of the PBMC population in humans.

By “pDC” herein is meant plasmacytoid dendritic cells and they representa subtype of dendritic cells found in the blood and peripheral lymphoidorgans. These cells express the surface markers CD123, BDCA-2(CD303) andBDCA-4(CD304) and HLA-DR, but do not express CD11c, CD14, CD3, CD20 orCD56, which distinguishes them from conventional dendritic cells,monocytes, T-cells, B cells and NK cells. As components of the innateimmune system, these cells express intracellular Toll-like receptors 7and 9, which enable the detection of viral and bacterial nucleic acids,such as ssRNA or CpG DNA motifs. Upon stimulation and subsequentactivation, these cells produce large amounts of Type I interferon(mainly IFN-α and IFN-β) and Type III interferon (e.g., IFN-γ), whichare critical pleiotropic anti-viral compounds mediating a wide range ofeffects. By generating a large number of type I interferon, cytokinesand chemokines, plasmacytoid dendritic cells are widely involved in thebody's innate and adaptive immune responses. They can regulate NK cells,T cells, B cells and other cells involved in immune response intensity,duration, and response mode, thus play a very important function intumor, infection and autoimmune disease. (Liu Y J. IPC: professionaltype 1 interferon-producing cells and plasmacytoid dendritic cellprecursors. Annu Rev Immunol. 2005; 23:275-306. Gilliet M, Cao W, Liu YJ. Plasmacytoid dendritic cells: sensing nucleic acids in viralinfection and autoimmune diseases. Nat Rev Immunol. 2008 August; 8(8):0.594-606).

By “mDC” herein is meant myeloid dendritic cells and they represent asubtype of circulating dendritic cells found in blood and peripherallymphoid organs. These cells express the surface markers CD11c, CD1a,HLA-DR and either BDCA-1 (CD1c) or BDCA-3 (CD141). They do not expressBDCA-2 or CD123, which distinguishes them from pDC. mDC also do notexpress CD3, CD20 or CD56. As components of the innate immune system,mDC express Toll-like receptors (TLR), including TLR2, 3, 4, 5, 6 and 8,which enable the detection of bacterial and viral components. Uponstimulation and subsequent activation, these cells are the most potentantigen presenting cells to activate antigen-specific CD4 as well as CD8T cells. In addition, mDCs has the ability to produce large amounts ofIL-12 and IL23, which is critical for the induction of Th1-mediated orTh17 cell-mediated immunity.

Study found that many solid tumors such as breast cancer and head andneck cancer, ovarian cancer has pDC's invasion (Treilleux I, Blay J Y,Bendriss-Vermare N et al. Dendritic cell infiltration and prognosis ofearly stage breast cancer. Clin Cancer Res 2004; 10:7466-7474. HartmannE, Wollenberg B, Rothenfusser S et al. Identification and functionalanalysis of tumor-infiltrating plasmacytoid dendritic cells in head andneck cancer. Cancer Res 2003; 63:6478-6487. Zou W P, Machelon V,Coulomb-L'Hermin A, et al. Stromal-derived factor-1 in human tumorsrecruits and alters the function of plasmacytoid precursor dendriticcells. Nat Med 2001; 7:1339-1346) and factors secreted by tumor cellsinhibit DC maturation. (Gabrilovich D I, Corak J, Ciemik I F et al.Decreased antigen presentation by dendritic cells in patients withbreast cancer. Clin Cancer Res 1997; 3:483-490. Bell D, Chomarat P,Broyles D et al. In breast carcinoma tissue, immature dendritic cellsreside within the tumor, whereas mature dendritic cells are located inperitumoral areas. J Exp Med 1999; 190:1417-1425. Menetrier-Caux C,Montmain G, Dieu M C et al. Inhibition of the differentiation ofdendritic cells from CD34 (+) progenitors by tumor cells: role ofinterleukin-6 and macrophage colony-stimulating factor. Blood 1998;92:4778-4791). These immature DC cells did not play a role in promotinganti-tumor immunity. By contrast, DCs within the tumor microenvironmentpromote tumor growth by inhibiting antitumor immunity and by promotingangiogenesis. There is evidence that Toll-like receptor 7 agonistImiquimod, and Toll-like receptor 9 agonist CpG drugs can stimulate pDCwithin the tumor microenvironment to inhibit tumor development. (DummerR, Urosevic M, Kempf W et al. Imiquimod in basal cell carcinoma: howdoes it work? Br J Dermatol 2003; 149:57-58. Miller R L, Gerster J F,Owens M L et al Imiquimod applied topically: a novel immune responsemodifier and new class of drag. Int J Immunopharmacol 1999; 21:1-14.Hofmann M A, Kors C, Audring H et al Phase 1 evaluation ofintralesionally injected TLR9-agonist PF-3512676 in patients with basalcell carcinoma or metastatic melanoma. J Immunother 2008; 31:520-527).

Natural killer (NK) cells are a type of cytotoxic lymphocyte thatconstitutes a major component of the immune system. NK cells are asubset of peripheral blood lymphocytes defined by the expression of CD56or CD 16 and the absence of the T cell receptor (CD3). They recognizeand kill transformed cell lines without priming in an MHC-unrestrictedfashion. NK cells play a major role in the rejection of tumors and cellsinfected by viruses. The process by which an NK cell recognizes a targetcell and delivers a sufficient signal to trigger target lysis isdetermined by an array of inhibitory and activating receptors on thecell surface. NK discrimination of self from altered self involvesinhibitory receptor recognition of MHC-I molecules and non-MHC ligandslike CD48 and Clr-1b. NK recognition of infected or damaged cells(altered self) is coordinated through stress induced ligands (e.g.,MICA, MICE, Rae1, H60, Mult1) or vitally encoded ligands (e.g., m157,hemaglutinin) recognized by various activating receptors, includingNKG2D, Ly49H and NKp46/Ncr1.

NK cells represent the predominant lymphoid cell in the peripheral bloodfor many months after allogeneic or autologous stem cell transplant andthey have a primary role in immunity to pathogens during this period(Reittie et al (1989) Blood 73: 1351-1358; Lowdell et al (1998) BoneMarrow Transplant 21: 679-686). The role of NK cells in engraftment,graft-versus-host disease, anti-leukemia activity and post-transplantinfection is reviewed in Lowdell (2003) Transfusion Medicine 13:399-404.

Human NK cells mediate the lysis of tumor cells and virus-infected cellsvia natural cytotoxicity and antibody-dependent cellular cytotoxicity(ADCC).

Human NK cells are controlled by positive and negative cytolyticsignals. Negative (inhibitory) signals are transduced by C-lectin domaincontaining receptors CD94/NKG2A and by some Killer Immunoglobulin-likeReceptors (KIRs). The regulation of NK lysis by inhibitory signals isknown as the “missing self” hypothesis in which specific HLA-class Ialleles expressed on the target cell surface ligate inhibitory receptorson NK cells. The down-regulation of HLA molecules on tumor cells andsome vitally infected cells (e.g. CMV) lowers this inhibition below atarget threshold and the target cells may become susceptible to NKcell-mediated lysis if the target cells also carry NK-priming andactivating molecules. TLR7, TLR8 or TLR9 agonists can activate both mDCand pDCs to produce type I IFNs and express costimulatory molecules suchas GITR-ligand, which subsequently activate NK cells to produce IFN-gand potently promote NK cell killing function.

Inhibitory receptors fall into two groups, those of the Ig-superfamilycalled Killer Immunoglobulin-like Receptors (KIRs) and those of thelectin family, the NKG2, which form dimers with CD94 at the cellsurface. KIRs have a 2- or 3-domain extracellular structure and bind toHLA-A, -B or -C. The NKG2/CD94 complexes ligate HLA-E.

Inhibitory KIRs have up to 4 intracellular domains which contain ITIMsand the best characterized are KIR2DL1, KIR2DL2 and KIR2DL3 which areknown to bind HLA-C molecules. KIR2DL2 and KIR2DL3 bind the group 1HLA-C alleles while KIR2DL1 binds to group 2 alleles. Certainleukemia/lymphoma cells express both group 1 and 2 HLA-C alleles and areknown to be resistant to NK-mediated cell lysis.

With regards to positive activating signals, ADCC is thought to bemediated via CD 16, and a number of triggering receptors responsible fornatural cytotoxicity have been identified, including CD2, CD38, CD69,NKRP-I, CD40, B7-2, NK-TR, NKp46, NKp30 and NKp44. In addition, severalKIR molecules with short intracytoplasmic tails are also stimulatory.These KIRs (KIR2DS1, KIR2DS2 and KIR2DS4) are known to bind to HLA-C;their extracellular domains being identical to their related inhibitoryKIRs. The activatory KIRs lack the ITIMs and instead associate with DAP12 leading to NK cell activation. The mechanism of control of expressionof inhibitory versus activatory KIRs remains unknown.

Several reports have described the expression of TLRs in mouse or humancancer or cancer cell lines. For example, TLR1 to TLR6 are expressed bycolon, lung, prostate, and melanoma mouse tumor cell lines (Huang B, etal. Toll-like receptors on tumor cells facilitate evasion of immunesurveillance. Cancer Res. 2005; 65(12):5009-5014), TLR3 is expressed inhuman breast cancer cells (Salaun B, Coste I, Rissoan M C, Lebecque S J,Renno T. TLR3 can directly trigger apoptosis in human cancer cells. JImmunol. 2006; 176(8):4894-4901), hepatocarcinoma and gastric carcinomacells express TLR2 and TLR4 (Huang B, et al. Listeria monocytogenespromotes tumor growth via tumor cell toll-like receptor 2 signaling.Cancer Res. 2007; 67(9):4346-4352), and TLR9 (Droemann D, et al. Humanlung cancer cells express functionally active Toll-like receptor 9.Respir Res. 2005; 6:1.) and TLR4 (He W, Liu Q, Wang L, Chen W, Li N, CaoX. TLR4 signaling promotes immune escape of human lung cancer cells byinducing immunosuppressive cytokines and apoptosis resistance. MolImmunol. 2007; 44(11):2850-2859.) are expressed by human lung cancercells. TLR7 and TLR8 are found in tumor cells of human lung cancer(Cherfils-Vicini J, Platonova S, Gillard M, Laurans L, Validire P,Caliandro R, Magdeleinat P, Mami-Chouaib F, Dieu-Nosjean M C, Fridman WH, Damotte D, Sautès-Fridman C, Cremer I. J. Clin Invest. 2010; 120(4):1285-1297).

TLR are a family of proteins that sense a microbial product and/orinitiates an adaptive immune response. TLRs activate a dendritic cell(DC). TLRs are conserved membrane spanning molecules containing anectodomain of leucine-rich repeats, a transmembrane domain and anintracellular TIR (Toll/interleukin receptor) domain. TLRs recognizedistinct structures in microbes, often referred to as “PAMPs” (pathogenassociated molecular patterns). Ligand binding to TLRs invokes a cascadeof intracellular signaling pathways that induce the production offactors involved in inflammation and immunity.

In some embodiments, the immunotherapeutic is a TLR7 and/or TLR8agonist. TLR7 and TLR8 are phylogenetically and structurally related.TLR7 is selectively expressed by human pDCs and B cells. TLR8 ispredominantly expressed mDCs, monocytes, macrophages and myeloidsuppressor cells. TLR7-specific agonists activate plasmacytoid DCs(pDCs) to produce large amounts of type 1 IFNs and expressing highlevels of costimulatory molecules that promote activation of T cells, NKcells, B cells and mDCs. TLR8-specific agonists activate myeloid DCs,monocytes, macrophages or myeloid-derived suppressor cells to producelarge amounts of type 1 IFN, IL-12 and IL-23, and express high levels ofMHC class I, MHC class II and costimulatory molecules that promote theactivation of antigen specific CD4 and CD8+ T cells.

In some embodiments, the immunotherapeutic is a TLR7 and/or TLR8 agonistthat is represented by the structure of Formula (I):

wherein dashed line represents bond or absence of bond;X is S or —NR₁, R₁ is -W₀-W₁-W₂-W₃-W₄,W₀ is a bond, alkyl alkenyl, alkynyl, alkoxy, or -alkyl-S-alkyl-,W₁ is a bond, —O—, or —NR₂—, wherein R₂ is hydrogen, alkyl or alkenyl,W₂ is a bond, —O—, —C(O)—, —C(S)—, or —S(O)₂—,W₃ is a bond, —NR₃—, wherein R₃ is hydrogen, alkyl or alkenyl,W₄ is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, aryl,aryloxy, heteroaryl, or heterocyclyl, each of which is optionallysubstituted by one or more substituents selected from the groupconsisting of hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl,aryl, heteroaryl, heterocyclyl. —NH₂, nitro, -alkyl-hydroxyl,-alkyl-aryl, -alkyl-heteroaryl, -alkyl-heterocyclyl, —O—R₄, —O-alkyl-R₄,-alkyl-O—R₄, —C(O)—R₄, -alkyl-C(O)—R₄, -alkyl-C(O)—O—R₄, —C(O)—O—R₄,—S—R₄, —S(O)₂—R₄, —NH—S(O)₂—R₄, -alkyl-S—R4, -alkyl-S(O)₂—R₄, —NHR₄,—NR₄R₄, —NH-alkyl-R₄, halogen, —CN, —NO₂, and —SH, wherein R₄ isindependently hydrogen, alkyl, alkenyl, -alkyl-hydroxyl, aryl,heteroaryl, heterocyclyl, or haloalkyl;Z is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryl, haloalkyl,heteroaryl, heterocyclyl, each of which can be optionally substituted byone or more substituents selected from the group consisting of hydroxyl,alkoxy, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl,halogen, cyano, nitro, —N(R₅)₂, -alkoxy-alkyl, -alkoxy-alkenyl,—C(O)-alkyl, —C(O)—O-alkyl, —O—C(O)-alkyl, —C(O)—N(R₅)₂, aryl,heteroaryl, —CO-aryl, and —CO-heteroaryl, wherein each R₅ isindependently hydrogen, alkyl, haloalkyl, -alkyl-aryl, or-alkyl-heteroaryl;R is hydrogen, alkyl, alkoxy, haloalkyl, halogen, aryl, heteroaryl,heterocyclyl, each of which is optionally substituted by one or moresubstituents selected from the group consisting of hydroxyl, alkoxy,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl,—NH₂, nitro, -alkyl-hydroxyl, -alkyl-aryl, -alkyl-heteroaryl,-alkyl-heterocyclyl. —O—R₄, —O-alkyl-R₄, -alkyl-O—R₄, —C(O)—R₄,—C(O)—NH—R₄, —C(O)—NR₄R₄, -alkyl-C(O)—R₄, -alkyl-C(O)—O—R₄, —C(O)—O—R₄,—O—C(O)—R₄, —S—R₄, —C(O)—S—R₄, —S—C(O)—R₄, —S(O)₂—R₄, —NH—S(O)₂—R₄,-alkyl-S—R₄, -alkyl-S(O)₂—R₄, —NHR₄, —NR₄R₄, —NH-alkyl-R₄, halogen, —CN,and —SH, wherein R₄ is independently hydrogen, alkyl, alkenyl, alkoxy,-alkyl-hydroxyl, aryl, heteroaryl, heterocyclyl, or haloalkyl;n is 0, 1, 2, 3, or 4;Y is —NR₆R₇, —CR₆R₇R₈, or -alkyl-NH₂, each of which can be optionallysubstituted by one or more substituents selected from the groupconsisting of hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, —NH₂, halogen,—N(R₅)₂, -alkoxyalkyl, -alkoxy-alkenyl, —C(O)-alkyl, —C(O)—O-alkyl,—C(O)—N(R₅)₂, aryl, heteroaryl, —CO-aryl, and —CO-heteroaryl.wherein R₆, R₇ and R₈ are independently hydrogen, alkyl, alkenyl,alkoxy, alkylamino, dialkylamino, alkylthio, arylthio, -alkyl-hydroxyl,-alkyl-C(O)—O—R₉, -alkyl-C(O)—R₉, or -alkyl-O—C(O)—R₉, wherein each R₅is independently hydrogen, alkyl, haloalkyl, -alkyl-aryl, or-alkyl-heteroaryl, wherein R₉ is hydrogen, alkyl, alkenyl, halogen, orhaloalkyl;X and Z taken together may optionally form a (5-9)-membered ring;or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, X of Formula (I) is S.

In some embodiments, X of Formula (I) is —NR₁, R₁ is alkyl, -alkyl-W₄,-alkyl-O—W₄, -alkyl-NH—C(O)—W₄, -alkoxy-NH—C(O)—W₄,-alkyl-NH—C(O)—NH—W₄, -alkoxy-NH—C(O)—NH—W₄, -alkyl-S(O)₂—W₄, or-alkyl-NH—C(S)—W₄, wherein W₄ is defined above.

In some embodiments, Z of Formula (I) is hydrogen, alkyl, alkoxy, aryl,heteroaryl, haloalkyl, each of which is optionally substituted by one tothree substituents selected from the group consisting of hydroxyl,alkyl, aryl, heteroaryl, heterocyclyl, cyano, -alkoxy-alkyl, nitro, and—N(R₅)₂, wherein each R₅ is independently hydrogen, alkyl, haloalkyl,-alkyl-aryl, or -alkyl-heteroaryl.

In some embodiments, Y of Formula (I) is —NH₂, -alkyl-NH₂, each of whichis optionally substituted by one to three substituents selected from thegroup consisting of alkyl, alkoxy, alkenyl, and alkynyl.

In some embodiments, n of Formula (I) is 1 or 2.

In some embodiments, R of Formula (I) is aryl or heteroaryl each ofwhich is optionally substituted by one to three substituents selectedfrom the group consisting of hydroxyl, alkoxy, -alkyl-hydroxyl, —O—R₄,—O-alkyl-R₄, -alkyl-O—R₄, —C(O)—R₄, —C(O)—NH—R₄, —C(O)—NR₄R₄,-alkyl-C(O)—R₄, -alkyl-C(O)—O—R₄, —C(O)—O—R₄, —O—C(O)—R₄, —S—R₄,—C(O)—S—R₄, —S—C(O)—R₄, —S(O)₂—R₄, —NH—S(O)₂—R₄, -alkyl-S—R₄,-alkyl-S(O)₂—R₄, —NHR₄, —NR₄R₄, —NH-alkyl-R₄, halogen, —CN, and —SH,wherein R is independently hydrogen, alkyl, alkenyl, alkoxy,-alkyl-hydroxyl, aryl, heteroaryl, heterocyclyl, or haloalkyl.

In some embodiments, the immunotherapeutic is a TLR7 and/or TLR8 agonistthat is selected from Table 2. The compounds in Table 2 are describedand characterized in more details in U.S. Pat. No. 4,689,338, U.S. Pat.No. 5,389,640, U.S. Pat. No. 5,226,575, U.S. Pat. No. 6,110,929, U.S.Pat. No. 6,194,425, U.S. Pat. No. 5,352,784. U.S. Pat. No. 6,331,539,U.S. Pat. No. 5,482,936, U.S. Pat. No. 6,451,810, WO2002/46192,WO2002/46193, WO2002/46194, US2004/0014779 and US2004/0162309.

TABLE 2 Representative TLR7 and/or TLR8 Agonists Name Structure2-propylthiazolo[4,5- c]quinolin-4-amine (CL075)

1-(2-methylpropyl)-1H- imidazo[4,5-c]quinolin-4- amine (Imiquimod)

4-amino-2- (ethoxymethyl)-a,a-di- methyl-1H-imidazo[4,5-c]quinoline-1-ethanol (Resiquimod)

1-(4-amino-2- ethylaminomethylimidazo- [4,5-c]quinolin-1-yl)-2-methylpropan-2-ol (Gardiquimod)

N-[4-(4-amino-2-ethyl- 1H-imidazo[4,5- c]quinolin-1-yl)butyl-]methanesulfonamide (CM001)

7-allyl-7,8-dihydro-8- oxo-guanosine (Loxoribine)

4-amino-2-ethoxymethyl- aa-dimethyl-6,7,8,9- tetrahydro-1h-imidazo[4,5-c]quinoline- 1-ethanol ol

4-amino-aa-dimethyl-2- methoxyethyl-1h- imidazo[4,5-c]quinoline-1-ethanol

1-(2-(3- (benzyloxy)propoxy)ethyl)- 2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4- amine

N-[4-(4-amino-2-butyl- 1H-imidazo[4,5- c][1,5]naphthyridin-1-yl)butyl]-n′-butylurea

N1-[2-(4-amino-2-butyl- 1H-imidazo[4,5-c][1,5] naphthyridin-1-yl)ethyl]-2-amino-4- methylpentanamide

N-(2-{2-[4-amino-2-(2- methoxyethyl)-1H- imidazo[4,5-c]quinolin-1-yl]ethoxy}ethyl)-n′- phenylurea

1-(2-amino-2- methylpropyl)-2- (ethoxymethyl)-1H-imidazo[4,5-c]quinolin- 4-amine

l-{4-[(3,5- dichlorophenyl)sulfonyl] butyl}-2-ethyl- 1H-imidazo[4,5-c]quinolin-4-amine

N-(2-{2-[4-amino-2- (ethoxymethyl)-1H- imidazo[4,5- c]quinolin-1-yl]ethoxy}ethyl)-n′- cyclohexylurea

N-{3-[4-amino-2- (ethoxymethyl)-1H- imidazo[4,5-c]quinolin-1-yl]propyl}- n′-(3- cyanophenyl)thiourea

N-[3-(4-amino-2-butyl- 1H-imidazo[4,5- c]quinolin-1-yl)-2,2-dimethylpropyl] benzamide

2-butyl-1-[3- (methylsulfonyl)propyl]- 1H-imidazo[4,5-c]quinolin-4-amine

N-{2-[4-amino-2- (ethoxymethyl)-1H- imidazo[4,5- c]quinolin-1-yl]-1,1-dimethylethyl}-2- ethoxyacetamide

1-[4-amino-2- ethoxymethyl-7-(pyridin- 4-yl)-1H- imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2- ol

1-[4-amino-2- (ethoxymethyl)-7- (pyridin-3-yl)-1H-imidazo[4,5-c]quinolin- 1-yl]-2-methylpropan-2- ol

N-{3-[4-amino-1-(2- hydroxy-2- methylpropyl)-2- (methoxyethyl)-1H-imidazo[4,5-c]quinolin-7- yl]phenyl}methane- sulfonamide

1-[4-amino-7-(5- hydroxymethylpyridin-3- yl)-2-(2- methoxyethyl)-1H-imidazo[4,5-c]quinolin-1- yl]-2-methylpropan-2-ol

3-[4-amino-2- (ethoxymethyl)-7- (pyridin-3-yl)-1H-imidazo[4,5-c]quinolin- 1-yl]propane-1,2-diol

1-[2-(4-amino-2- ethoxymethyl-1H- imidazo[4,5- c]quinolin-1-yl)-1,1 -dimethylethyl]-3- propylurea

1-[2-(4-amino-2- ethoxymethyl-1H- imidazo[4,5- c]quinolin-1-yl)-1,1-dimethylethyl]-3- cyclopentylurea

1-[(2,2-dimethyl-1,3- dioxolan-4-yl)methyl]-2- (ethoxymethyl)-7-(4-hydroxymethylphenyl)- 1H-imidazo[4,5-c] quinolin-4-amine

4-[4-amino-2- ethoxymethyl-1-(2- hydroxy-2- methylpropyl)-1H-imidazo[4,5-c]quinolin- 7-yl]-N- methoxy-N- methylbenzamide

2-ethoxymethyl-N1- isopropyl-6,7,8,9- tetrahydro-1H- imidazo[4,5-c]quinoline-1,4-diamine

1-[4-amino-2-ethyl-7- (pyridin-4-yl)-1H- imidazo[4,5-c]quinolin-l-yl]-2- methylpropan-2-ol

N-[4-(4-amino-2-ethyl- 1H-imidazo[4,5- c]quinolin-1- yl)butyl]methane-sulfonamide

N-[4-(4-amino-2-butyl- 1H-imidazo[4,5- c][1,5]naphthyridin-1-yl)butyl]-n′- cyclohexylurea

3M-34240

3M-052

3M-854A

Preferably in some embodiments, the immunotherapeutic is Resiquimod orImiquimod.

In some embodiments, the immunotherapeutic is a TLR modulator (e.g.,TLR7 and/or TLR8 agonist) that is represented by structure of Formula(II):

wherein V is —NR₆R₇, wherein each of R₆ and R₇ is independentlyhydrogen, alkyl, alkenyl, alkoxy, alkylamino, dialkylamino, alkylthio,arylthio, -alkyl-hydroxyl, -alkyl-C(O)—O—R₉, -alkyl-C(O)—R₉, or-alkyl-O—C(O)—R₉, wherein R₉ is hydrogen, alkyl, alkenyl, hydrogen, orhaloalkyl;R₁₀ and R₁₁ are independently hydrogen, alkyl, alkenyl, aryl, haloalkyl,heteroaryl, heterocyclyl, or cycloalkyl, each of which is optionallysubstituted by one or more substituents selected from the groupconsisting of hydroxyl, alkoxy, alkyl, alkenyl, alkynyl, halogen.—N(R₅)₂, -alkoxyalkyl, -alkoxy-alkenyl, —C(O)-alkyl, —C(O)—O-alkyl,—C(O)—N(R₅)₂, aryl, heteroaryl, —CO-aryl, and —CO-heteroaryl, whereineach R₅ is independently hydrogen, alkyl, haloalkyl, -alkyl-aryl, or-alkyl-heteroaryl,or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the immunotherapeutic is a TLR modulator (e.g.,TLR7 and/or TLR8 agonist) that is represented by structure of Formula(III):

wherein

is a double bond or a single bond; R₂ and R₃ are independently selectedfrom H and lower alkyl, or R₂ and R₃ are connected to form a saturatedcarbocycle having from 3 to 7 ring members; one of R₇ and R₈ is

and the other is hydrogen; R₄ is —NR_(c)R_(d) or —OR₁₀; R_(c) and R_(d)are lower alkyl, where the alkyl is optionally substituted with one ormore —OH; R₁₀, is alkyl, where the alkyl is optionally substituted withone or more —OH; Z is C and

is a double bond, or Z is N and

is a single bond; R_(a) and R_(b) are independently selected from H,alkyl, alkenyl, alkynyl, and R_(e), wherein the alkyl is optionallysubstituted with one or more —OR₁₀, or R_(e), R_(e) is selected from—NH₂, —NH(alkyl), and —N(alkyl)₂, R₁ is absent when

is a double bond, or when

is a single bond. N₁—R₁ and one of R_(a) or R_(b) are connected to forma saturated, partially unsaturated, or unsaturated heterocycle having5-7 ring members and the other of R_(a) or R_(b) may be hydrogen orabsent as necessary to accommodate ring unsaturation; and at least oneof the following A-D applies: A) R₇ is not hydrogen B) R₈ is nothydrogen and at least one of R_(a) and R_(b) is not hydrogen; C) Z is N;or D) N₁—R₁ and one of R_(a) or R_(b) are connected to form a saturated,partially unsaturated, or unsaturated heterocycle having 5-7 ringmembers. US 20140088085A1, the disclosure of which is incorporated byreferences in its entirety.

In some embodiments, R₇ of the compound of Formula (III) is

Additionally, at least one of R_(a) and R_(b) is not hydrogen in thecompound of Formula (III), or, for example, one of R_(a) and R_(b) isalkyl and the other of R_(a) and R_(b) is hydrogen. Further, the alkylof Formula (III) is substituted with R_(e). In a different embodiment,both R_(a) and R_(b) are alkyl or, one of R_(a) and R_(b) is R_(e) andthe other R_(a) and R_(b) is hydrogen. For example, R₈ of formula (III)is not hydrogen.

In some alternative embodiments, N₁ and one of R_(a) or R_(b) of Formula(III) are connected to form a saturated, partially unsaturated, orunsaturated heterocycle having 5-7 ring members and the other of R_(a)or R_(b) is hydrogen, or absent as necessary to accommodate ringunsaturation, where the ring is a 5 membered ring, or, for example, thering is:

In some embodiments, at least one of R₂ and R₃ in the compound ofFormula (III) is not hydrogen, or, for example, R₂ and R₃ are connectedto form a saturated carbocycle, where the saturated carbocycle iscyclopropyl. Alternatively, Z is N in the compound of Formula (III).

In some embodiments, the TLR agonist or modulator has the structure ofFormula (IV):

wherein R₄ is selected from —NR_(c)R_(d) and —OR₁₀; R_(c) and R_(d) arelower alkyl, where the alkyl is optionally substituted with one or more—OH; R₁₀ is alkyl, where the alkyl is optionally substituted with one ormore —OH, R_(f) and R_(g) are lower alkyl or R_(f) and R_(g) togetherwith the nitrogen atom to which they are attached form a saturatedheterocyclic ring having 4-6 ring members. For example, R_(f) and R_(g)in the compound of Formula (IV), together with the nitrogen atom towhich they are attached form a saturated heterocyclic ring, where theheterocyclic ring is pyrrolidine.

In some alternative embodiments, R₄ of either Formula (III) or Formula(IV) is —OR₁₀, where R₁₀ is alkyl or is ethyl. In another embodiment, R₄of either Formula (III) or Formula (IV) is —NR_(c)R_(d), where both arealkyl or both are propyl. Moreover, in certain embodiments, at least oneof R_(c) or R_(d) is alkyl substituted with one —OH and at least one ofR_(c) and R_(d) is

and the remaining R_(c) or R_(d) is propyl.

In some alternative embodiments, the TLR is a a compound selected from

Alternatively, the compound is selected from

In some alternative embodiments, the TLR agonist ound is either

In some alternative embodiments, the TLR agonist is a compound selectedfrom

In some alternative embodiments, the TLR agonist is

In some alternative embodiments, the TLR agonist is a compound selectedfrom:

In some embodiments, the immunotherapeutic is a TLR modulator (e.g.,TLR7 and/or TLR8 agonist) that is represented by structure of Formula(V):

and metabolites, solvates, tautomers, and prodrugs thereof, wherein:

Y is CF₂CF₃, CF₂CF₂R⁶ or an aryl or heteroaryl ring, wherein said aryland heteroaryl rings are substituted with one or more groupsindependently selected from alkenyl, alkynyl, Br, CN, OH, NR⁶R⁷,C(═O)R⁸, NR⁶SO₂R⁷, (C₁-C₆ alkyl)amino, R⁶OC(═O)CH═CH₂—, SR⁶ and SO₂R⁶,and wherein the aryl and heteroaryl rings are optionally furthersubstituted with one or more groups independently selected from F, Cl,CF₃, CF₃O—, HCF₂O—, alkyl, heteroalkyl and ArO—;

R¹, R³ and R⁴ are independently selected from H, alkyl, alkenyl,alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryland heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl₅, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶,C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OC(̂O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶,

or R³ and R⁴ together with the atom to which they are attached form asaturated or partially unsaturated carbocyclic ring, wherein thecarbocyclic ring is optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN,OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino,CH₃OCH₂O—, R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶:

R² and R⁸ are independently selected from H, OR⁶, NR⁶R⁷, alkyl, alkenyl,alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryland heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br₅ I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶,C(═O)OR⁶, OC(═O)R⁶, C(̂O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶;

R^(5a), R^(5b), and R^(5c) are independently H, F, Cl, Br, I₅ OMe₅ CH₃,CH₂F₅ CHF₂ or CF₃; and

R⁶ and R⁷ are independently selected from H₅ alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl andheteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, C(═O)R⁶,C(═O)OR⁶, OC(O)R⁶, C(O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OC(̂O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶,

or R⁶ and R⁷ together with the atom to which they are attached form asaturated or partially unsaturated heterocyclic ring, wherein saidheterocyclic ring is optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN,OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino,CH₃OCH₂O—, R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶. In certainembodiments, R¹, R³ and R⁴ are each hydrogen. In certain embodiments,R^(5a), R^(5b) and R^(5c) are each hydrogen. WO 2007024612 A2, thedisclosure of which is incorporated by reference in its entirety.

In some embodiments of the compound of Formula (V), R² is OR⁶. In someembodiments, R⁶ is alkyl, such as (1-4C)alkyl. In particularembodiments, R⁶ is ethyl.

In some embodiments of the compound of Formula (V), R² is NR⁶R⁷. In someembodiments, R⁶ and R⁷ are independently H, alkyl, such as (1-6C)alkyl,or heteroalkyl, such as (1-4C)alkoxy(2-4C)alkyl. In particularembodiments, R⁶ and R⁷ are independently H, ethyl, propyl, orCH₂CH₂OCH₃. In some embodiments of the compound of Formula V, Y is aryl,such as phenyl. In some embodiments, the aryl is substituted withC(═O)R⁸, such as in para-R⁸C(═O)phenyl. In some embodiments, R⁸ is OR⁶,NR⁶R⁷ or heterocycloalkyl. In some embodiments, R⁶ and R⁷ areindependently H or alkyl, such as (1-6C)alkyl. In some otherembodiments, R⁶ and R⁷ together with the nitrogen atom to which they areattached form a 4-6 membered azacycloalkyl ring, such as pyrrolidinyl.In some embodiments, Y is

In some embodiments of the compound of Formula (V), Y is CF₂CF₃.

In some embodiments, the immunotherapeutic is a TLR modulator (e.g.,TLR8 agonist) that is represented by structure of formula (VI):

and metabolites, solvates, tautomers, and pharmaceutically acceptableprodrugs and salts thereof, wherein:

Z is H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl. OR⁶ or NR⁶R⁷, wherein said alkyl,alkenyl, alkynyl; heteroalkyl, cycloalkyl, heterocycloalkyl, aryl andheteroaryl are optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl₃ Br, I, CN,OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, CC₁-C₆alkyl)amino,CH₃OCH₂O—, R⁶OCC═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶;

R¹, R², R³ and R⁴ are independently selected from H, alkyl, alkenyl,alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryland heteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, CC═O)R⁶,C(═O)OR⁶, OC(═O)R⁶, CC═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OCC═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶,

or R¹ and R² together with the atom to which they are attached form asaturated or partially unsaturated carbocyclic ring, wherein saidcarbocyclic ring is optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN,OR⁶, NR⁶R⁷, C(═O)R⁶, CC═O)OR⁶, OC(═O)R⁶, CC═O)NR⁶R⁷, (C₁-C₆ alkyl)amino,CH₃OCH₂O—, R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶;

or R³ and R⁴ together are oxo;

each R⁵ is independently selected from H, F, Cl, Br, I, OMe, CH₃, CH₂F,CHF₂, CF₃ and CF₂CF₃;

R⁶ and R⁷ are independently selected from H, alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, andheteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R⁷, CC═O)R⁶,C(=0)0R⁶, 0C(=0)R⁶, CC═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶;

or R⁶ and R⁷ together with the atom to which they are attached form asaturated or partially unsaturated heterocyclic ring, wherein theheterocyclic ring is optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN,OR⁶, NR⁶R⁷, CC═O)R⁶, C(=0)0R⁶, 0C(=0)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino,CH₃OCH₂O—, R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶; and n is O, 1, 2, 3or 4. WO2007040840A2, the disclosure of which is incorporated byreference in its entirety.

In some embodiments, the immunotherapeutic is a TLR modulator (e.g.,TLR8 agonist) that is represented by structure of Formula (VI):

and metabolites, solvates, tautomers, and pharmaceutically acceptablesalts and prodrugs thereof, wherein.

Z is H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, OR⁶ or NR⁶R⁷, wherein the alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl andheteroaryl are optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN,OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino,CH₃OCH₂O—, R⁶OCC═O)CH═CH₂, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶;

R¹, R², R³ and R⁴ are independently selected from H, alkyl, alkenyl,alkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryland heteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br, I₉ CN, OR⁶, NR⁶R⁷, C(═O)R⁶,C(═O)OR₆, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino, CH₃OCH₂O—,R⁶OCC═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶,

or R¹ and R² together with the atom to which they are attached form asaturated or partially unsaturated carbocyclic ring, wherein saidcarbocyclic ring is optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN,OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆alkyl)amino,CH₃OCH₂O—, R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶,

or R³ and R⁴ together are oxo;

R⁵ is H, F, Cl, Br, I, OMe, CH₃, CH₂F, CHF₂, CF₃ or CF₂CF₃;

R⁶ and R⁷ are independently selected from H, alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, andheteroaryl, wherein said alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, and heteroaryl areoptionally substituted with one or more groups independently selectedfrom alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN, OR⁶, NR⁶R₇, C(═O)R⁶,C(=0)0R⁶, 0C(=0)R⁶, C(O)NR⁶R⁷, (C₁-C₆ alkyl)amino₅ CH₃OCH₂O—,R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶;

or R⁶ and R⁷ together with the atom to which they are attached form asaturated or partially unsaturated heterocyclic ring, wherein saidheterocyclic ring is optionally substituted with one or more groupsindependently selected from alkyl, alkenyl, alkynyl, F, Cl, Br, I, CN,OR⁶, NR⁶R⁷, C(═O)R⁶, C(═O)OR⁶, OC(═O)R⁶, C(═O)NR⁶R⁷, (C₁-C₆ alkyl)amino,CH₃OCH₂O—, R⁶OC(═O)CH═CH₂—, NR⁶SO₂R⁷, SR⁶ and SO₂R⁶, and

n is O, 1, 2, 3 or 4.

In some embodiments, Z is OR⁶. In some embodiments, R⁶ is alkyl, such as(1-6C)alkyl. In particular embodiments, R⁶ is ethyl, propyl, isopropylor isobutyl.

In some embodiments, Z is NR⁶R⁷. In some embodiments, R⁶ and R⁷ areindependently H or alkyl, such as (1-6C)alkyl. In some embodiments, R⁶and R⁷ are ethyl. In some embodiments, n is O or 1.

In some embodiments, R⁵ is CF₂CF₃. In certain embodiments, R³ is H oralkyl, such as (1-4C)alkyl, and R⁴ is H. In certain embodiments, R isalkyl, such as (1-4C)alkyl. In some embodiments, R is methyl. In otherparticular embodiments, R³ is H. In some embodiments, R is H or alkyl,such as (1-4C)alkyl and R is H. In some embodiments, R¹ is alkyl. Insome embodiments, R¹ is methyl. In some particular embodiments, R¹ is H.

In some embodiments, the activating moiety is a TLR7 and/or TLR8 agonistthat is represented by structure of Formula (XV):

wherein ring A represents a 6-10 membered aromatic carbocyclic ring or a5-10 membered heteroaromatic ring;R represents a halogen atom, an alkyl group, a hydroxyalkyl group, ahaloalkyl group, an alkoxy group, a hydroxyalkoxy group, a haloalkoxygroup, amino group, an alkylamino group, a dialkylamino group, or a 4-7membered cyclic group containing in the ring 1-2 hetero atoms selectedfrom 1-2 nitrogen atoms and optionally 0-1 oxygen atom or 0-1 sulfuratom;n represents an integer of 0-2, and when n is 2, the Rs may be the sameor different;Z¹ represents a substituted or unsubstituted alkylene group or asubstituted or unsubstituted cycloalkylene group;X² represents oxygen atom, sulfur atom, SO₂, NR⁵, CO, CONR⁵, NR⁵CO,SO₂NR⁵, NR⁵SO₂, NR⁵CONR⁶ or NR⁵CSNR⁶ (in which R⁵ and R⁶ are eachindependently hydrogen atom, a substituted or unsubstituted alkyl group,or a substituted or unsubstituted cycloalkyl group);Y¹, Y² and Y³ represent each independently a single bond or an alkylenegroup;X¹ represents oxygen atom, sulfur atom, SO₂, NR⁴ (wherein R⁴ is hydrogenatom or an alkyl group) or a single bond;R² represents hydrogen atom, a substituted or unsubstituted alkyl group,a substituted or unsubstituted alkenyl group, a substituted orunsubstituted alkynyl group or a substituted or unsubstituted cycloalkylgroup; andR¹ represents hydrogen atom, hydroxy group, an alkoxy group, analkoxycarbonyl group, a haloalkyl group, a haloalkoxy group, asubstituted or unsubstituted aryl group, a substituted or unsubstitutedheteroaryl group or a substituted or unsubstituted cycloalkyl group. Thelinker is linked to one of the possible linking site of the angonist,such as to —NH₂.

In some embodiments, R¹ represents hydrogen, hydroxyl, or a C₁-C₆alkoxy, C₂-C₅alkoxycarbonyl, C₁-C₆ haloalkyl, C₁-C₆ haloalkoxy, C₆-C₁₀aryl, C₅-C₁₀heteroaryl or C₁-C₈ cycloalkyl group, each group beingoptionally substituted by one or more substituents independentlyselected from halogen, hydroxyl, a C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆alkoxy, C₁-C₆haloalkoxy, C₂-C₅ alkoxycarbonyl, amino (NH₂), (mono)-C₁-C₆alkylamino and (di)-C₁-C₆ alkylamino group;

Y¹ represents a single bond or C₁-C₆ alkylene;X¹ represents a single bond, an oxygen, sulphur atom, sulphonyl (SO₂) orNR³;Z¹ represents a C₂-C₆ alkylene or C₃-C₈ cycloalkylene group, each groupbeing optionally substituted by at least one hydroxyl;X² represents NR⁴;Y² represents a single bond or C₁-C₆ alkylene;Y³ represents a single bond or C₁-C₆ alkylene;n is an integer 0, 1 or 2;R represents halogen or a C₁-C₆ alkyl, C₁-C₆ hydroxyalkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy, C₁-C₆ hydroxyalkoxy, C₁-C₆ haloalkoxy, amino(NH₂), (mono)-C₁-C₆ alkylamino, (di)-C₁-C₆ alkylamino group or aC₃-C₈saturated heterocyclic ring containing a ring nitrogen atom andoptionally one or more further heteroatoms independently selected fromnitrogen, oxygen and sulphur, the heterocyclic ring being optionallysubstituted by one or more substituents independently selected fromhalogen, hydroxyl, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₅ alkylcarbonyland C₂-C₅alkoxycarbonyl;R² represents hydrogen or a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl orC₃-C₈ cycloalkyl group, each group being optionally substituted by oneor more substituents independently selected from halogen, hydroxyl or aC₁-C₆ alkoxy, a C₂-C₁₀ acyloxy, group selected from a C₂₋₅alkylcarbonyloxy group, a C₂-C₅ alkenylcarbonyloxy group, a C₂-C₅alkynylcarbonyloxy group, a C₆-C₉ arylcarbonyloxy group and aC₅-C₉heteroarylcarbonyloxy group, each of which acyloxy groups may beoptionally substituted by one or more substituents independentlyselected from halogen, hydroxyl, C₁-C₃ alkoxy and phenyl providing thatthe total number of carbon atoms in the acyloxy group does not exceed10, amino (NH₂), (mono)-C₁-C₆ alkylamino, (di)-C₁-C₆ alkylamino groupand a C₃-C₈ saturated heterocyclic ring containing a ring nitrogen atomand optionally one or more further heteroatoms independently selectedfrom nitrogen, oxygen and sulphur, the heterocyclic ring in turn beingoptionally substituted by one or more substituents independentlyselected from halogen, hydroxyl, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy,C₂-C₅alkylcarbonyl and C₂-C₅ alkoxycarbonyl group;R³ represents hydrogen or C₁-C₆ alkyl;R⁴ represents CO₂R⁵, SO₂R⁵, COR⁵, SO₂NR⁶R⁷ and CONR⁶R⁷;R⁵ independently represents(i) 3- to 8-membered heterocyclic ring containing 1 or 2 heteroatomsselected from a ring group NR⁸, S(O)_(m) or oxygen, the 3- to 8-memberedheterocyclic ring being optionally substituted by one or moresubstituents independently selected from halogen, hydroxyl or a C₁-C₆alkyl and C₁-C₆ alkoxy group, or(ii) a C₆-C₁₀ aryl or C₅-C₁₀ heteroaryl group, each of which may beoptionally substituted by one or more substituents independentlyselected from halogen, cyano, C₁-C₆ alkyl, C₁-C₃ haloalkyl, carboxyl,S(O)_(m)R⁹, OR¹⁰, CO₂R¹⁰, SO₂NR¹⁰R¹¹, CONR¹⁰R¹¹, NR¹⁰R¹¹, NR¹⁰SO₂R⁹,NR¹⁰CO₂R⁹, NR¹⁰COR⁹, or(iii) a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl or C₃-C₈ cycloalkylgroup, each of which may be optionally substituted by one or moresubstituents independently selected from halogen, CN, C₃-C₈cycloalkyl,S(O)_(p)R¹², OR¹³, COR¹³, CO₂R¹³, SO₂NR¹³R¹⁴, CONR¹³R¹⁴, NR¹³R¹⁴,NR¹³SO₂R¹², NR¹³CO₂R¹², NR¹³COR¹², NR¹³SO₂R¹² or a C₆-C₁₀ aryl or C₅-C₁₀heteroaryl group or a heterocyclic ring, the latter three groups may beoptionally substituted by one or more substituents independentlyselected from C₁-C₆ alkyl (optionally substituted by hydroxy, C₁-C₆alkoxy, C₁-C₆ alkoxycarbonyl, amino, C₁-C₆ alkylamino, di-C₁-C₆alkylamino, NH₂C(O)—, C₁-C₆ alkylNHC(O), di-C₁-C₆ alkyl NC(O),—OCH₂CH₂OH, pyrrolidinyl, pyrrolidinylcarbonyl, furanyl, piperidyl,methylpiperidyl or phenyl), C₂-C₆ alkenyl (optionally substituted byphenyl), halogen, hydroxy, cyano, carboxy, amino, C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, NH₂C(O)—, C₁-C₆ alkyl NHC(O)—, di-C₁-C₆ alkylNC(O), C₁-C₆ alkoxycarbonyl, C₁-C₆ alkylsulphonyl, C₁-C₆alkylcarbonylamino, C₁-C₆ alkylcarbonylmethylamino, phenyl (optionallysubstituted by hydroxy, fluoro or methyl), pyrrolidinyl, pyridyl,piperidinyl, benzothiazolyl or pyrimidinyl;R⁶ represents hydrogen or a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl group or heterocyclic ring, each of which may beoptionally substituted by one or more substituentsindependently-selected from halogen, hydroxyl, oxo, cyano, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, OR¹⁵, S(O)_(q)R¹⁵,CO₂R¹⁶, COR¹⁶, NR¹⁶R¹⁷, CONR¹⁶R¹⁷, NR¹⁶COR¹⁷, NR¹⁶CO₂R¹⁵, SO₂NR¹⁶R¹⁷,NR¹⁶SO₂R¹⁵, or a C₆-C₁₀ aryl or C₅-C₁₀ heteroaryl group or heterocyclicring, the latter three groups being optionally substituted by one ormore substituents independently selected from, C₁-C₆ alkyl, C₃-C₈cycloalkyl, halogen, S(O)_(q)R¹⁵, CO₂R¹⁶, COR¹⁶, hydroxy or cyano; andR⁷ represents hydrogen, a C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, orC₃-C₈ cycloalkyl group, each group may be optionally substituted by oneor more substituents independently selected from halogen,C₃-C₈cycloalkyl, a C₆-C₁₀ aryl or C₅-C₁₀ heteroaryl group, carboxy,cyano, OR¹⁵, hydroxy or NR¹⁸R¹⁹, orR⁶ and R⁷ together with the nitrogen atom to which they are attachedfowl a 3- to 8-membered saturated or partially saturated heterocyclicring, optionally containing further heteroatoms or heterogroups selectedfrom nitrogen. S(O)_(m) or oxygen, the heterocyclic ring, may beoptionally substituted by one or more substituents independentlyselected from halogen, hydroxyl, carboxyl, cyano, OR²⁰, NR²¹R²²,S(O)_(q)R²³, COR²⁴, CO₂R²⁴, NR²⁴R²⁵, CONR²⁴R²⁵, NR²⁴COR²⁵, NR²⁴CO₂R²³,SO₂NR²⁴R²⁵, NR²⁴SO₂R²³, C₆-C₁₀ aryl, C₅-C₁₀ heteroaryl group,heterocyclic ring, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl orC₃-C₈cycloalkyl group, the latter seven groups being optionallysubstituted by one or more substituents independently selected fromhalogen, hydroxyl, oxo, cyano, OR²⁰, S(O)_(q)R²³, COR²⁴, CO₂R²⁴,NR²⁴R²⁵, CONR²⁴R²⁵, NR²⁴CO₂R²³, NR²⁴COR²⁵, SO₂NR²⁴R²⁵, NR²⁴SO₂R²³, aheterocyclic ring or a C₆-C₁₀ aryl or C₅-C₁₀ heteroaryl group, thelatter three groups being optionally substituted by one or moresubstituents independently selected from C₁-C₆ alkyl, halogen, hydroxyor cyano;R⁸ represents hydrogen, CO₂R²⁶, COR²⁶, SO₂R²⁶, C₁-C₆ alkyl or C₃-C₆cycloalkyl group, each group may be optionally substituted by one ormore substituents independently selected from halogen, hydroxyl, andNR²⁷R²⁸;R¹⁰, R¹¹, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²¹, R²², R²⁶, R²⁷ or R²⁸ eachindependently represents hydrogen, and a C₁-C₆ alkyl or C₃-C₆ cycloalkylgroup;R²⁴ and R²⁵ each independently represents hydrogen, and a C₁-C₆ alkyl orC₃-C₆ cycloalkyl group; orR²⁴ and R²⁵ together with the nitrogen atom to which they are attachedform a 3- to 8-membered saturated or partially saturated heterocyclicring, optionally containing further heteroatoms or heterogroups selectedfrom nitrogen, S(O)_(m) or oxygen;R⁹, R¹², R¹⁵ and R²³ represent C₁-C₆ alkyl or C₃-C₆ cycloalkyl;R¹³ and R¹⁴ are defined as for R⁶ and R⁷ respectively;R²⁰ represents a C₁-C₆ alkyl optionally substituted by one or moresubstituents independently selected from halogen, hydroxyl or OR²³;m, p, q and r each independently represent an integer 0, 1 or 2; andA represents a C₆-C₁₀ aryl or C₅-C₁₂ heteroaryl group. SeeWO2008004948A1, U.S. Pat. No. 8,138,172, and U.S. Pat. No. 8,575,180 thedisclosure of which is incorporated by reference.

In some embodiments, the activating moiety is a TLR7 and/or TLR8 agonisthaving the structure of:

wherein R is Me or H.

In some embodiments, the activating moiety is a TLR7 and/or TLR8 agonisthaving the structure of:

In some embodiments, the activating moiety is a TLR7 and/or TLR8 agonisthaving the structure of Formula (XVI):

wherein: R¹ is independently H, —C(O)R³, or a racemic, L-, or D-aminoacid group—C(O)CHNH₂R⁴, wherein R³ is a substituted or unsubstituted alkyl, and R⁴is H, or a substituted or unsubstituted alkyl;R² is H, O, OR⁵, or N(R⁶)₂, wherein R⁵ is independently H or alkyl, andwherein R⁶ is independently H, substituted or unsubstituted alkyl,cycloalkyl, or together with nitrogen forms a substituted orunsubstituted heterocycloalkyl ring; and wherein if R is —OH, at leastone of the R groups is a racemic, L-, or D-amino acid group—C(O)CHNH₂R⁴. See U.S. Pat. No. 6,924,271, the disclosure of which isincorporated by reference in its entirety.

In some embodiments, at least one of the R¹ groups is a racemic. L-, orD-amino acid group —C(O)CHNH₂R⁴, wherein R⁴ is a substituted orunsubstituted alkyl, and wherein the remaining R¹ groups are H; R² isOR⁵ or N(R⁶)₂, wherein R⁵ is independently selected from H or alkyl, andwherein R is independently H, substituted or unsubstituted alkyl,cycloalkyl, or together with nitrogen forms a substituted orunsubstituted heterocycloalkyl ring.

In some embodiments, at least one of the R¹ groups is a L-amino acidgroup —C(O)CHNH₂R⁴, wherein R⁴ is a substituted or unsubstituted alkyl,and wherein the remaining R¹ groups are H; R² is OR⁵ or N(R⁶)₂, whereinR⁴ is a substituted alkyl, and wherein R⁶ is independently H orsubstituted or unsubstituted alkyl.

In some embodiments, at least one of the R¹ groups is a L-amino acidgroup —C(O)CHNH₂R, wherein R⁴ is —CH(CH₃)₂, and wherein the remaining R¹groups are H; and R² is OH.

In some embodiments, the TLR7 and/or agonist is selected from the groupconsisting of:

In some embodiments, the activating moiety is a TLR7 and/or TLR8 agonisthaving the structure of:

wherein:

each R¹ is H, or a substituted or unsubstituted alkyl, alkenyl, oralkynyl, which may be interrupted by one or more O, S, or N heteroatoms,or a substituted or unsubstituted aryl or heteroaryl;

R² is H, OH, SH, halo, or a substituted or unsubstituted alkyl, alkenyl,or alkynyl, which may be interrupted by one or more O, S, or Nheteroatoms, or a substituted or unsubstituted —O-(alkyl), —O-(aryl),—O-(heteroaryl), —S-(alkyl), —S-(aryl), —S-(heteroaryl), aryl, orheteroaryl;

R³ is H, OH, or SH, or a substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, heteroaryl, —O-(alkyl), —O-(aryl), —O-(heteroaryl),—S-(alkyl), —S-(aryl), —S-(heteroaryl), —NH(alkyl), —NH(aryl),—NH(heteroaryl), —NH(R⁴)(alkyl), —NH(R⁴)(aryl), or —NH(R⁴)(heteroaryl),wherein R⁴ is a substituted or unsubstituted alkyl;

X is O or S;

V is H, halo, OH, OR⁴, SH, SR⁴, or a substituted or unsubstituted alkylor aryl; and

Z is H, halo, OH, OR⁴, SH, or SR⁴. See U.S. Pat. No. 7,576,068, thedisclosure of which is incorporated by reference in its entirety.

In some embodiments, the activating moiety is a TLR7 and/or TLR8 agonisthaving the structure of Formula (XVIII):

wherein:Y—Z is —CR⁴R⁵—, —CR⁴R⁵—CR⁴R⁵—, —C(O)CR⁴R⁵—, —CR⁴R⁵C(O)—, —NR⁸C(O)—,—C(O)NR⁸—, CR⁴R⁵S(O)₂—, or —CR⁵═CR⁵—;L¹ is —NR⁸—, —O—, —S—, —N(R⁸)C(O)—, —S(O)₂—, —S(O)—C(O)N(R⁸)—,—N(R⁸)S(O)₂—, —S(O)₂N(R⁸)— or a covalent bond:R¹ is alkyl, substituted alkyl, haloalkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, heteroalkyl, substituted heteroalkyl,carbocyclyl, substituted carbocyclyl, carbocyclylalkyl, substitutedcarbocyclylalkyl, heterocyclyl, substituted heterocyclyl,heterocyclylalkyl, or substituted heterocyclylalkyl, arylalkyl,substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl,carbocyclylheteroalkyl, substituted carbocyclylheteroalkyl,heterocyclylheteroalkyl, substituted heterocyclylheteroalkyl,arylheteroalkyl, substituted arylheteroaryl, heteroarylheteroalkyl, orsubstituted heteroarylheteroalkyl;X¹ is alkylene, substituted alkylene, heteroalkylene, substitutedheteroalkylene, alkenylene, substituted alkenylene, alkynylene,substituted alkynylene, carbocyclylene, substituted carbocyclylene,heterocyclylene, substituted heterocyclylene, —NR⁸—, —O—, —C(O)—,—S(O)—, —S(O)₂—, or a bond;D is carbocyclyl, substituted carbocyclyl, heterocyclyl or substitutedheterocyclyl wherein said carbocyclyl, substituted carbocyclyl,heterocyclyl or substituted heterocyclyl is substituted with one or two-L²-NR⁶R⁷; orD is a heterocyclyl, substituted heterocyclyl, heteroaryl or substitutedheteroaryl wherein said heterocyclyl, substituted heterocyclyl,heteroaryl or substituted heteroaryl comprises one to four nitrogenatoms;each L² is independently alkylene, substituted alkylene, heteroalkylene,substituted heteroalkylene, or a covalent bond;each R³ is independently halogen, cyano, azido, nitro, alkyl,substituted alkyl, hydroxyl, amino, heteroalkyl, substitutedheteroalkyl, alkoxy, haloalkyl, haloalkoxy, —CHO, —C(O)OR⁸, —S(O)R⁸,—S(O)₂R⁸; —C(O)NR⁹R¹⁰, —N(R⁹)C(O)R⁸, carbocyclyl, substitutedcarbocyclyl, carbocyclylalkyl, substituted carbocyclylalkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, —S(O)₂NR⁹R¹⁰,—N(R⁹)S(O)₂R⁸, —N(R⁹)S(O)₂OR¹⁰, —OS(O)₂NR⁹R¹⁰;n is 0, 1, 2, 3, 4 or 5;R⁴ and R⁵ are each independently H, alkyl, substituted alkyl, haloalkyl,heteroalkyl, substituted heteroalkyl, carbocyclyl, substitutedcarbocyclyl, carbocyclylalkyl, substituted carbocyclylalkyl,heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substitutedheterocyclylalkyl, arylalkyl, substituted arylalkyl, heteroarylalkyl,substituted heteroarylalkyl, carbocyclylheteroalkyl, substitutedcarbocyclylheteroalkyl, heterocyclylheteroalkyl, substitutedheterocyclylheteroalkyl, arylheteroalkyl, substituted arylheteroalkyl,heteroarylheteroalkyl, or substituted heteroarylheteroalkyl, cyano,azido, OR⁸, —C(O)H, —C(O)R⁸, —S(O)R⁸, —S(O)₂R⁸, —C(O)OR⁸, or—C(O)NR⁹R¹⁰; orR⁴ and R⁵, taken together with the carbon to which they are bothattached, form a carbocycle, substituted carbocycle, heterocycle orsubstituted heterocycle; orR⁴ and R⁵, when on the same carbon atom, taken together with the carbonto which they are attached are —C(O)— or —C(NR⁸)—; ortwo R⁴ or two R⁵ on adjacent carbon atoms when taken together with thecarbons to which they are attached form a 3 to 6 membered carbocycle,substituted carbocycle, heterocycle or substituted heterocycle;R⁶ and R7 are each independently H, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, haloalkyl,heteroalkyl, substituted heteroalkyl, carbocyclyl, substitutedcarbocyclyl, carbocyclylalkyl, substituted carbocyclylalkyl,heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substitutedheterocyclylalkyl, arylalkyl, substituted arylalkyl, heteroarylalkyl,substituted heteroarylalkyl, carbocyclylheteroalkyl, substitutedcarbocyclylheteroalkyl, heterocyclylheteroalkyl, substitutedheterocyclylheteroalkyl, arylheteroalkyl, substituted arylheteroalkyl,heteroarylheteroalkyl, or substituted heteroarylheteroalkyl, —C(O)H,—C(O)R⁸, —S(O)R⁸, —S(O)₂R⁸, —C(O)OR⁸, or —C(O)NR⁹R¹⁰, S(O)₂NR⁹R¹⁰; orR⁶ and R⁷, taken together with the nitrogen to which they are bothattached, form a substituted or unsubstituted heterocycle, which maycontain one or more additional heteroatoms selected from N, O, P, or S;orR⁷ taken together with L², and the N to which they are both attached,forms a substituted or unsubstituted 3 to 8 membered heterocycle whichmay contain one or more additional heteroatoms selected from N, O, S, orP;R⁸ is H, alkyl, substituted alkyl, haloalkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, heteroalkyl, substitutedheteroalkyl, carbocyclyl, substituted carbocyclyl, carbocyclylalkyl,substituted carbocyclylalkyl, heterocyclyl, substituted heterocyclyl,heterocyclylalkyl, substituted heterocyclylalkyl, arylalkyl, substitutedarylalkyl, heteroarylalkyl, substituted heteroarylalkyl,carbocyclylheteroalkyl, substituted carbocyclylheteroalkyl,heterocyclylheteroalkyl, substituted heterocyclylheteroalkyl,arylheteroalkyl, substituted arylheteroalkyl, heteroarylheteroalkyl, orsubstituted heteroarylheteroalkyl; andR⁹ and R¹⁰ are each independently H, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, haloalkyl,heteroalkyl, substituted heteroalkyl, carbocyclyl, substitutedcarbocyclyl, carbocyclylalkyl, substituted carbocyclylalkyl,heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substitutedheterocyclylalkyl, arylalkyl, substituted arylalkyl, heteroarylalkyl,substituted heteroarylalkyl, carbocyclylheteroalkyl, substitutedcarbocyclylheteroalkyl, heterocyclylheteroalkyl, substitutedheterocyclylheteroalkyl, arylheteroalkyl, substituted arylheteroalkyl,heteroarylheteroalkyl, or substituted heteroarylheteroalkyl; orR⁹ and R¹⁰, taken together with the nitrogen to which they are bothbonded, form a substituted or unsubstituted heterocycle;wherein each substituted alkyl, substituted alkenyl, substitutedalkynyl, substituted heteroalkyl, substituted carbocyclyl, substitutedcarbocyclylalkyl, substituted heterocyclyl, substitutedheterocyclylalkyl, substituted arylalkyl, substituted heteroarylalkyl,substituted carbocyclylheteroalkyl, substituted heterocyclylheteroalkyl,substituted arylheteroalkyl, substituted heteroarylheteroalkyl,substituted alkylene, substituted heteroalkylene, substitutedalkenylene, substituted alkynylene, substituted carbocyclylene, orsubstituted heterocyclylene is independently substituted with one tofour substituents selected from the group consisting of -halogen, —R,—O⁻, ═O, —OR, —SR, —S⁻, —NR₂, —N(+)R₃, ═NR, —C(halogen)₃, —CR(halogen)₂,—CR₂(halogen), —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃,—NRC(═O)R, —NRC(═O)OR, —NRC(═O)NRR, —C(═O)NRR, —C(═O)OR, —OC(═O)NRR,OC(═O)OR, —C(═O)R, —S(═O)₂OR, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NR, —S(═O)R,—NRS(═O)₂R, —NRS(═O)₂NRR, —NRS(═O)₂OR, —OP(═O)(OR)₂, —P(═O)(OR)₂,—P(O)(OR)(O)R, —C(═O)R, —C(═S)R, —C(═O)OR, —C(═S)OR, —C(═C)SR, —C(═S)SR,—C(═O)NRR, —C(═S)NRR, —C(═NR)NRR, and —NRC(═NR)NRR; wherein each R isindependently H, alkyl, cycloalkyl, aryl, arylalkyl, or heterocyclyl.See US 20100143301 A1, the disclosure of which is incorporated byreference in its entirety.

In some embodiments, the activating moiety is a TLR7 and/or TLR8 agonisthaving the structure of:

wherein:

L¹ is —NH— or —O—;

R¹ is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl,heterocyclylalkyl, substituted heterocyclylalkyl, carbocyclylalkyl orsubstituted carbocyclylalkyl;each of R⁴ and R⁵ independently is H or C₁-C₆ alkyl or R⁴ and R⁵ takentogether with the carbon to which they are attached is —C(O)—;X¹ is C₁-C₆ alkylene. C₁-C₆ heteroalkylene or C₁-C₆ substitutedheteroalkylene;D is phenyl, biphenyl or pyridinyl, wherein said phenyl, biphenyl orpyridinyl is substituted with -L²-NR⁶R⁷; orD is pyridinyl, piperidinyl, piperazinyl or1,2,3,4-tetrahydroisoquinolinyl;n is 0 or 1;R³ is halogen, cyano, alkyl, carbocyclyl, carbocyclylalkyl, haloalkyl,—C(O)OR⁶, —C(O)NR⁹R¹⁰ or —CHO;L² is C₁-C₆ alkylene or a covalent bond;each of R⁶ and R⁷ independently is H, alkyl, or heteroaryl; orR⁶ and R⁷ taken together with the nitrogen to which they are attachedform a substituted or unsubstituted 4-6 membered heterocycle comprising0 to 2 heteroatoms selected from N, O or S.

In some embodiments, the activating moiety is a TLR7 and/or TLR8 agonisthaving the structure of:

C. Amount of Immunotherapeutics in the Therapeutic Combinations

In another aspect, the present invention provides a therapeuticcombination comprising a target therapeutic and an immunotherapeutic inan amount that is suitable for the combination therapy treatment ofdiseases such as tumors and cancers.

In some embodiments, the immunotherapeutic is of an amount that iscapable of: (1) inducing IFN-α in a enriched human blood DCs; (2)inducing TNF-α in a enriched human blood DCs; and/or (3) inducingIL-12-α in a enriched human blood DCs.

Methods for measuring the activity of the immunotherapeutics are: 1) anassay to measure cytokines release from human dendritic cell stimulatedby immunotherapy; 2) an assay to detect antibody dependent cell mediatedcytotoxicity enhanced by immunotherapy; and 3) an efficacy study in atumor model treated by immunotherapy.

In some embodiments, the immunotherapeutic (e.g. resiquimod or itsanalogues) is adminstered, either orally or intravenously using oralformulation or intravenous formulation, of an amount so that the localconcentration of the immunotherapeutics (e.g. near or at the tumor siteof a solid tumor) is between about 0.005 μg/ml to about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12 μg/ml (all inclusive).

The local concentration of the immunotherapeutics (e.g. near or at thetumor site of a solid tumor) can measured using methods known in theart, such as measuring the tissue or serum concentration.

Local effective concentration of therapeutic agent is depended on itsabsorption from various routes, tissue distribution, and metabolismprocess, and plasma pharmacokinetics of agent and tissue concentrationcould be measured routinely using methods known in the art.

In some embodiments, the immunotherapeutic is adminstered of an amountso that the local concentration of the immunotherapeutics (e.g. near orat the tumor site of a solid tumor) is between about 0.05 μg/ml, 0.1μg/ml, 0.15 μg/ml, 0.2 μg/ml, 0.3 μg/ml, or 0.4 μg/ml, to about 0.5μg/ml (all inclusive).

In some embodiments, the subject is administered an oral formulationcomprising the immunotherapeutic (e.g. resiquimod or its analogues) in adose of between about 0.0005 mg/kg, 0.0006 mg/kg, 0.0007 mg/kg, 0.0008mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg,0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01mg/kg, or 0.015 mg/kg, to about 0.02 mg/kg (all inclusive), two timesper week. In some embodiments, the subject is administered an oralformulation comprising the immunotherapeutic (e.g. resiquimod or itsanalogues) in a dose of between about 0.0005 mg/kg, to about 0.0006mg/kg, 0.0007 mg/kg, 0.0008 mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0.002mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg,0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.015 mg/kg, or 0.02 mg/kg (allinclusive), two times per week.

In some embodiments, the subject is administered an oral formulationcomprising the immunotherapeutic (e.g. resiquimod or its analogues) in adose of less than or about 0.0005 mg/kg, 0.0006 mg/kg, 0.0007 mg/kg,0.0008 mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg,0.01 mg/kg, two times per week.

In some embodiments, the subject is administered an intravenousformulation comprising the immunotherapeutic (e.g. resiquimod or itsanalogues) in a dose of between about 0.0005 mg/kg, 0.0006 mg/kg, 0.0007mg/kg, 0.0008 mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0.002 mg/kg, 0.003mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg,0.009 mg/kg, 0.01 mg/kg, or about 0.015 mg/kg, to about 0.02 mg/kg(inclusive), weekly. In some embodiments, the subject is administered anintravenous formulation comprising the immunotherapeutic (e.g.resiquimod or its analogues) in a dose of between about 0.0005 mg/kg, toabout 0.0006 mg/kg, 0.0007 mg/kg, 0.0008 mg/kg, 0.0009 mg/kg, 0.001mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg,0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.015 mg/kg, or 0.02mg/kg (inclusive), weekly.

In some embodiments, the subject is administered a formulationcomprising the immunotherapeutic (e.g. resiquimod or its analogues) in adose of between about 0.0005 mg/kg, 0.0006 mg/kg, 0.0007 mg/kg, 0.0008mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg,0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01mg/kg, 0.02 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg,0.125 mg/kg, 0.15 mg/kg, 0.175 mg/kg, 0.2 mg/kg, 0.215 mg/kg, 0.25mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.25 mg/kg, 1.5 mg/kg, 1.75mg/kg, 2 mg/kg, 2.25 mg/kg, to about 2.5 mg/kg, all inclusive, twicedaily, once daily, once every two, three, four, five or six days, oronce, twice, or three times per week.

In some embodiments, the subject is administered a formulationcomprising the immunotherapeutic (e.g. resiquimod or its analogues) in adose of between about 0.0005 mg/kg, to about 0.0006 mg/kg, 0.0007 mg/kg,0.0008 mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg,0.01 mg/kg, 0.02 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg,0.125 mg/kg, 0.15 mg/kg, 0.175 mg/kg, 0.2 mg/kg, 0.215 mg/kg, 0.25mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.25 mg/kg, 1.5 mg/kg, 1.75mg/kg, 2 mg/kg, 2.25 mg/kg, or 2.5 mg/kg o, all inclusive, twice daily,once daily, once every two, three, four, five or six days, or once,twice, or three times per week.

In some embodiments, the subject is administered a formulationcomprising die immunotherapeutic (e.g. resiquimod or its analogues) in adose of less than or about 0.0005 mg/kg, 0.0006 mg/kg, 0.0007 mg/kg,0.0008 mg/kg, 0.0009 mg/kg, 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg,0.01 mg/kg, 0.02 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg,0.125 mg/kg, 0.15 mg/kg, 0.175 mg/kg, 0.2 mg/kg, 0.215 mg/kg, 0.25mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.25 mg/kg, 1.5 mg/kg, 1.75mg/kg, 2 mg/kg, 2.25 mg/kg, or 2.5 mg/kg, twice daily, once daily, onceevery two, three, four, five or six days, or once, twice, or three timesper week.

In some embodiments, the administration is orally, sublingually,intravenously, intramuscularly, subcutaneously, or intratumorally. Insome embodiments, the subject is administered a formulation comprisingthe immunotherapeutic (e.g. resiquimod or its analogues) in a dose of arange between any two doses selected from the following doses: about0.0005 mg/kg, 0.0006 mg/kg, 0.0007 mg/kg, 0.0008 mg/kg, 0.0009 mg/kg,0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.02 mg/kg,0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15mg/kg, 0.175 mg/kg, 0.2 mg/kg, 0.215 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75mg/kg, 1 mg/kg, 1.25 mg/kg, 1.5 mg/kg, 1.75 mg/kg, 2 mg/kg, 2.25 mg/kg,and 2.5 mg/kg.

In some embodiments, the method comprises administering to said subjectan intravenous formulation comprising said immunotherapeutic (e.g.resiquimod or its analogues) in a dose of between about from 0.0008mg/kg to about 0.0133 mg/kg, weekly.

In some embodiments, the subject is administered an intravenousformulation comprising the immunotherapeutic (e.g. resiquimod or itsanalogues) in a dose of less than or about 0.003 mg/kg, 0.004 mg/kg,0.005 mg/kg, or 0.006 mg/kg to about 0.007 mg/kg, weekly. For referencesregarding safe dosage of immunotherapeutics, see Jurk et al., NatureImmunology, Vol. 4, No. 6″499 (2002), and Pockros et al., J. Hepatology,47:174-182 (2007), the disclosure of which is incorporated by referencein their entirety.

III. Pharmaceutical Formulations and Administration

The present invention further relates to a pharmaceutical formulationcomprising a compound of the invention or a pharmaceutically acceptablesalt thereof, and one or more pharmaceutically acceptable carriers.

The compounds described herein including pharmaceutically acceptablecarriers such as addition salts or hydrates thereof can be delivered toa patient using a wide variety of routes or modes of administration.Suitable routes of administration include, but inhalation, transdermal,oral, rectal, transmucosal, intestinal and parenteral administration,including intramuscular, subcutaneous and intravenous injections.Preferably, the compounds of the invention comprising an antibody orantibody fragment as the targeting moiety are administered parenterally,more preferably intravenously.

As used herein, the terms “administering” or “administration” areintended to encompass all means for directly and indirectly delivering acompound to its intended site of action.

The compounds described herein, or pharmaceutically acceptable saltsand/or hydrates thereof, may be administered singly, in combination withother compounds of the invention, and/or in cocktails combined withother therapeutic agents. Of course, the choice of therapeutic agentsthat can be co-administered with the compounds of the invention willdepend, in part, on the condition being treated.

For example, when administered to patients suffering from a diseasestate caused by an organism that relies on an autoinducer, the compoundsof the invention can be administered in cocktails containing agents usedto treat the pain, infection and other symptoms and side effectscommonly associated with the disease. Such agents include, e.g.,analgesics, antibiotics, etc.

When administered to a patient undergoing cancer treatment, thecompounds may be administered in cocktails containing anti-cancer agentsand/or supplementary potentiating agents. The compounds may also beadministered in cocktails containing agents that treat the side-effectsof radiation therapy, such as anti-emetics, radiation protectants, etc.

Supplementary potentiating agents that can be co-administered with thecompounds of the invention include, e.g., tricyclic anti-depressantdrugs (e.g., imipramine, desipramine, amitriptyline, clomipramine,trimipramine, doxepin, nortriptyline, protriptyline, amoxapine andmaprotiline); non-tricyclic and anti-depressant drugs (e.g., sertraline,trazodone and citalopram); Ca+2 antagonists (e.g., verapamil,nifedipine, nitrendipine and caroverine); amphotericin; triparanolanalogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine);antihypertensive drugs (e.g., reserpine); thiol depleters (e.g.,buthionine and sulfoximine); and calcium leucovorin.

The active compound(s) of the invention are administered per se or inthe form of a pharmaceutical composition wherein the active compound(s)is in admixture with one or more pharmaceutically acceptable carriers,excipients or diluents. Pharmaceutical compositions for use inaccordance with the present invention are typically formulated in aconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries, which facilitateprocessing of the active compounds into preparations which, can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, and suspensions for oral ingestion by apatient to be treated. Pharmaceutical preparations for oral use can beobtained solid excipient, optionally grinding a resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired to obtain tablets or dragee cores. Suitable excipients are,in particular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations, which can be used orally, include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Injection isa preferred method of administration for the compositions of the currentinvention. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents may be added, such as the cross-linked polyvinyl pyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents, which increase the solubility of thecompounds to allow for the preparation of highly, concentratedsolutions. For injection, the agents of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hanks's solution, Ringer's solution, or physiological salinebuffer.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation or transcutaneous delivery (e.g.,subcutaneously or intramuscularly), intramuscular injection or atransdermal patch. Thus, for example, the compounds may be formulatedwith suitable polymeric or hydrophobic materials (e.g., as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude calcium carbonate, calcium phosate, various sugars, starches,cellulose derivatives, gelatin, and polymers such as polyethyleneglycols.

A preferred pharmaceutical composition is a composition formulated forinjection such as intravenous injection and includes about 0.01% toabout 100% by weight of the compound of the present invention, basedupon 100% weight of total pharmaceutical composition. The drug-ligandconjugate may be an antibody-cytotoxin conjugate where the antibody hasbeen selected to target a particular cancer.

In some embodiments, the pharmaceutical composition of the presentinvention further comprises an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is an anticanceragent.

In some embodiments, the additional anticancer agent is selected from anantimetabolite, an inhibitor of topoisomerase I and II, an alkylatingagent, a microtubule inhibitor, an antiandrogen agent, a GNRh modulatoror mixtures thereof.

In some embodiments, the additional therapeutic agent is achemotherapeutic agent.

By “chemotherapeutic agent” herein is meant a chemical compound usefulin the treatment of cancer. Examples are but not limited to:Gemcitabine, Irinotecan, Doxorubicin, 5-Fluorouracil, Cytosinearabinoside (“Ara-C”), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin,TAXOL, Methotrexate, Cisplatin, Melphalan, Vinblastine and Carboplatin.

In some embodiments, the second chemotherapeutic agent is selected fromthe group consisting of tamoxifen, raloxifene, anastrozole, exemestane,letrozole, imatanib, paclitaxel, cyclophosphamide, lovastatin, minosine,gemcitabine, cytarabine, 5-fluorouracil, methotrexate, docetaxel,goserelin, vincristine, vinblastine, nocodazole, teniposide etoposide,gemcitabine, epothilone, vinorelbine, camptothecin, daunorubicin,actinomycin D, mitoxantrone, acridine, doxorubicin, epirubicin, oridarubicin.

IV. Kits

In another aspect, the present invention provides kits containing thetherapeutic combinations provided herein and directions for using thetherapeutic combinations. The kit may also include a container andoptionally one or more vial, test tube, flask, bottle, or syringe. Otherformats for kits will be apparent to those of skill in the art and arewithin the scope of the present invention.

V. Medical Use

In another aspect, the present invention provides a method for treatinga disease condition in a subject that is in need of such treatment,comprising: administering to the subject a therapeutic combination orpharmaceutical composition comprising a therapeutically effective amountof the compound of the present invention or a pharmaceuticallyacceptable salt thereof, and a pharmaceutical acceptable carrier.

In addition to the compositions and constructs described above, thepresent invention also provides a number of uses of the combinations ofthe invention. Uses of the combinations of the current inventioninclude: killing or inhibiting the growth, proliferation or replicationof a tumor cell or cancer cell, treating cancer, treating apre-cancerous condition, preventing the multiplication of a tumor cellor cancer cell, preventing cancer, preventing the multiplication of acell that expresses an autoimmune antibody. These uses compriseadministering to an animal such as a mammal or a human in need thereofan effective amount of a compound of the present invention.

The combination of the current invention is useful for treating diseasessuch as cancer in a subject, such as a human being. Combinations anduses for treating tumors by providing a subject the composition in apharmaceutically acceptable manner, with a pharmaceutically effectiveamount of a composition of the present invention are provided.

By “cancer” herein is meant the pathological condition in humans that ischaracterized by unregulated cell proliferation. Examples include butare not limited to: carcinoma, lymphoma, blastoma, and leukemia. Moreparticular examples of cancers include but are not limited to: lung(small cell and non-small cell), breast, prostate, carcinoid, bladder,gastric, pancreatic, liver (hepatocellular), hepatoblastoma, colorectal,head and neck squamous cell carcinoma, esophageal, ovarian, cervical,endometrial, mesothelioma, melanoma, sarcoma, osteosarcoma, liposarcoma,thyroid, desmoids, chronic myelocytic leukemia (AML), and chronicmyelocytic leukemia (CML).

By “inhibiting” or “treating” or “treatment” herein is meant toreduction, therapeutic treatment and prophylactic or preventativetreatment, wherein the objective is to reduce or prevent the aimedpathologic disorder or condition. In one example, followingadministering of a compound of the present invention, a cancer patientmay experience a reduction in tumor size. “Treatment” or “treating”includes (1) inhibiting a disease in a subject experiencing ordisplaying the pathology or symptoms of the disease, (2) ameliorating adisease in a subject that is experiencing or displaying the pathology orsymptoms of the disease, and/or (3) affecting any measurable decrease ina disease in a subject or patient that is experiencing or displaying thepathology or symptoms of the disease. To the extent a compound of thepresent invention may prevent growth and/or kill cancer cells, it may becytostatic and/or cytotoxic.

By “therapeutically effective amount” herein is meant an amount of acompound provided herein effective to “treat” a disorder in a subject ormammal. In the case of cancer, the therapeutically effective amount ofthe drug may either reduce the number of cancer cells, reduce the tumorsize, inhibit cancer cell infiltration into peripheral organs, inhibittumor metastasis, inhibit tumor growth to certain extent, and/or relieveone or more of the symptoms associated with the cancer to some extent.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order. As used herein, the term “pharmaceutical combination”refers to a product obtained from mixing or combining activeingredients, and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound of Formula (1) and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound of Formula (1) and a co-agent, are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the activeingredients in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of three or more activeingredients.

In some embodiments, the diseases condition is tumor or cancer. In someembodiments, the cancer or tumor is selected from stomach, colon,rectal, liver, pancreatic, lung, breast, cervix uteri, corpus uteri,ovary, testis, bladder, renal, brain/CNS, head and neck, throat,Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, leukemia,melanoma, non-melanoma skin cancer, acute lymphocytic leukemia, acutemyelogenous leukemia, Ewing's sarcoma, small cell lung cancer,choriocarcinoma, rhabdomyosarcoma, Wilms' tumor, neuroblastoma, hairycell leukemia, mouth/pharynx, oesophagus, larynx, kidney cancer orlymphoma.

In some embodiments, the disease condition comprises abnormal cellproliferation, such as a pre-cancerous lesion.

The current invention is particularly useful for the treatment of cancerand for the inhibition of the multiplication of a tumor cell or cancercell in an animal. Cancer, or a precancerous condition, includes atumor, metastasis, or any disease or disorder characterized byuncontrolled cell growth, can be treated or prevented by administrationthe drug-ligand complex of the current invention. The compound deliversthe activating moiety to a tumor cell or cancer cell. In someembodiments, the targeting moiety specifically binds to or associateswith a cancer-cell or a tumor-cell-associated antigen. Because of itsclose proximity to the ligand, after being internalized, the activatingmoiety can be taken up inside a tumor cell or cancer cell through, forexample, receptor-mediated endocytosis. The antigen can be attached to atumor cell or cancer cell or can be an extracellular matrix proteinassociated with the tumor cell or cancer cell. Once inside the cell, thelinker is hydrolytically or enzymatically cleaved by a tumor-cell orcancer-cell-associated proteases, thereby releasing the activatingmoiety. The released activating moiety is then free to diffuse andinduce or enhance immune activity of immune cells or tumor cells. In analternative embodiment, the activating moiety is cleaved from thecompound tumor microenvironment, and the drug subsequently penetratesthe cell.

Representative examples of precancerous conditions that may be targetedby the compounds of the present invention, include: metaplasia,hyperplasia, dysplasia, colorectal polyps, actinic ketatosis, actiniccheilitis, human papillomaviruses, leukoplakia, lychen planus andBowen's disease.

Representative examples of cancers or tumors that may be targeted bycompounds of the present invention include: lung cancer, colon cancer,prostate cancer, lymphoma, melanoma, breast cancer, ovarian cancer,testicular cancer, CNS cancer, renal cancer, kidney cancer, pancreaticcancer, stomach cancer, oral cancer, nasal cancer, cervical cancer andleukemia. It will be readily apparent to the ordinarily skilled artisanthat the particular targeting moiety used in the compound can be chosensuch that it targets the activating moiety to the tumor tissue to betreated with the drug (i.e., a targeting agent specific for atumor-specific antigen is chosen). Examples of such targeting moiety arewell known in die art, examples of which include anti-Her2 for treatmentof breast cancer, anti-CD20 for treatment of lymphoma, anti-PSMA fortreatment of prostate cancer and anti-CD30 for treatment of lymphomas,including non-Hodgkin's lymphoma.

In some embodiments, the abnormal proliferation is of cancer cells.

In some embodiments, the cancer is selected from the group consistingof: breast cancer, colorectal cancer, diffuse large B-cell lymphoma,endometrial cancer, follicular lymphoma, gastric cancer, glioblastoma,head and neck cancer, hepatocellular cancer, lung cancer, melanoma,multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer,and renal cell carcinoma.

In some embodiments, the present invention provides a compound for usein killing a cell. The compound is administered to the cell in an amountsufficient to kill said cell. In an exemplary-embodiment, the compoundis administered to a subject bearing the cell. In a further exemplaryembodiment, the administration serves to retard or stop the growth of atumor that includes the cell (e.g., the cell can be a tumor cell). Forthe administration to retard the growth, the rate of growth of the cellshould be at least 10% less than the rate of growth beforeadministration. Preferably, the rate of growth will be retarded at least20% 30%, 40%, 50%, 60%, 70%, 80%, 90%, or completely stopped.

Additionally, the present invention provides a compound or apharmaceutical composition of the present invention for use as amedicament. The present invention also provides a compound or apharmaceutical composition for killing, inhibiting or delayingproliferation of a tumor or cancer cell, or for treating a diseasewherein TLR7 and/or TLR8 are implicated.

Effective Dosages

Pharmaceutical compositions suitable for use with the present inventioninclude compositions wherein the active ingredient is contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. Determination of an effective amount is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure herein.

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays. Target plasmaconcentrations will be those concentrations of active compound(s) thatare capable of inhibition cell growth or division. In preferredembodiments, the cellular activity is at least 25% inhibited. Targetplasma concentrations of active compound(s) that are capable of inducingat least about 30%, 50%, 75%, or even 90% or higher inhibition ofcellular activity are presently preferred. The percentage of inhibitionof cellular activity in the patient can be monitored to assess theappropriateness of the plasma drug concentration achieved, and thedosage can be adjusted upwards or downwards to achieve the desiredpercentage of inhibition.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a circulating concentration thathas been found to be effective in animals. The dosage in humans can beadjusted by monitoring cellular inhibition and adjusting the dosageupwards or downwards, as described above.

A therapeutically effective dose can also be determined from human datafor compounds which are known to exhibit similar pharmacologicalactivities. The applied dose can be adjusted based on the relativebioavailability and potency of the administered compound as comparedwith the known compound.

Adjusting the dose to achieve maximal efficacy in humans based on themethods described above and other methods as are well-known in the artis well within the capabilities of the ordinarily skilled artisan.

In some embodiments, the composition of the present invention isdelivered local or regional to a tumor located in the subject, deliveredsystemically, or delivered via intratumoral injection or by directinjection into tumor vasculature.

In the case of local administration, the systemic circulatingconcentration of administered compound will not be of particularimportance. In such instances, the compound is administered so as toachieve a concentration at the local area effective to achieve theintended result.

Therapeutic amounts of specific antibodies disclosed herein can also beadministered, as a component of the combination, with theimmunotherapeutics, either in a single mixture form, or separately. Insome embodiments, therapeutic amounts are amounts which eliminate orreduce the patient's tumor burden, or which prevent or reduce theproliferation of metastatic cells. The dosage will depend on manyparameters, including the nature of the tumor, patient history, patientcondition, the possible co-use of other oncolytic agents, and methods ofadministration. Methods of administration include injection (e.g.,parenteral, subcutaneous, intravenous, intraperitoneal, etc.) for whichthe antibodies are provided in a nontoxic pharmaceutically acceptablecarrier such as water, saline, Ringer's solution, dextrose solution, 5%human serum albumin, fixed oils, ethyl oleate, or liposomes. Typicaldosages may range from about 0.01 to about 20 mg/kg, such as from about0.1 to about 10 mg/kg. Other effective methods of administration anddosages may be determined by routine experimentation and are within thescope of this invention.

The therapeutically effective amount of the agents (disclosed herein)administered, when it is used for combination therapy, can varydepending upon the desired effects and the subject to be treated. Forexample, the subject can receive at least 1 mg/kg (such as 1 mg/kg to 20mg/kg, 2.5 mg/kg to 10 mg/kg, or 3.75 mg/kg to 5 mg/kg) intravenously ofeach antibody agent. The dosage can be administered in divided doses(such as 2, 3, or 4 divided doses per day), or in a single dosage.

In the method for combined administration, the agent may besimultaneously administered with the antibody used in the presentinvention, or the agent may be administered before or after theadministration of the antibody used in the present invention.

For other modes of administration, dosage amount and interval can beadjusted individually to provide plasma levels of the administeredcompound effective for the particular clinical indication being treated.For example, in one embodiment, a compound according to the inventioncan be administered in relatively high concentrations multiple times perday. Alternatively, it may be more desirable to administer a compound ofthe invention at minimal effective concentrations and to use a lessfrequent administration regimen. This will provide a therapeutic regimenthat is commensurate with the severity of the individual's disease.

Utilizing the teachings provided herein, an effective therapeutictreatment regimen can be planned which does not cause substantialtoxicity and yet is entirely effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration and the toxicity-profile of the selected agent.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES

The present invention is further exemplified, but not limited, by thefollowing and Examples that illustrate the preparation of the compoundsof the invention.

Example 1

Tumour Inoculation and Evaluation of Tumour Growth

Mice: Female 6-week-old BALB/c and C3H/HeN (C3H) mice were purchasedfrom Japan SLC (Hamamatsu, Japan). All procedures were reviewed andapproved by the Animal Care and Use Committee of the Tokyo Medical andDental University. The SCCVII (C3H-originated, 3×10⁵), or Colon26(BALB/c-originated, 5×10⁵) parental cells were injected subcutaneously(s.c.) into the shaved right flank of syngeneic mice and tumour volumeswere evaluated. In experiments examining the effects of anti-PDL1 (MIH5)mAb with TLRL, 200 ug of anti-PDL1 mAb or 200 ug of anti-PDL1 mAbmixture with TLRL or control rat IgG was injected i.p. three times aweek after tumour inoculation. Tumor volumes were measured along threeorthogonal axes (x, y, and z) and calculated as tumor volume=(xyz)/2, ifa mouse lose more than 20% of body weight or is very sick and cannot getto adequate food or water, it will be removed from the study andeuthanized. (FIGS. 1 and 2)

Example 2

Enrichment of Human Dendritic Cells (DCs) from PBMC

Human PBMC was prepared from Buffy coats obtained from healthy volunteerdonors by Ficoll centrifugation. Dendritic cells were enriched by usingnegative depletion with magnetic beads (Miltenyi Biotec Inc. San Diego,Calif.) with mixture of anti-CD3, CD19, CD20, CD14, and CD16 antibodiesfrom human PBMC. The enrichment of DCs was stained with goat anti-mouseFITC (lineages), HLA-DR-APCCy7, CD123-BV421 and CD11C-APC. The stainedcells were analyzed on BD LSR Fortessa (BD Biosciences). The anti-CD3,CD4, CD11C, CD19, CD14, CD16, CD123 monoclonal antibody were purchasedfrom BD Biosciences, CA or Biolegend, San Diego, Calif.

Stimulation of Enriched Human DCs and Cytokines Expression

1-2×10⁵ enriched DCs were plated in a 96-well plate in 100 μl media, 100μl diluted stimulators (including TLRL were add to the plate andcultured for 20-22 h in 37° C. incubator. The supernatant were collectedand human IFN-α, IL-12(p70) and TNF-α were analyzed by ELISA (MabtechAB, Sweden).

FIGS. 3A-3G depict analysis of cytokine production by enriched human DCsfrom three healthy donors. Enriched human DCs were plated in a 96-wellplate and cultured with allogeneic untreated (medium) or treateddifferent concentration of TLRL directly for 20-22 h in 37° C.incubator. The supernatant were collected and human IFN-α, IL-12(p70)and TNF-α were analyzed by ELISA. Data are given as mean±SD oftriplicate cultures. Three independent experiments from three healthydonors were performed (Donor 1: FIG. 3A, Donor 2: FIG. 3B-D; Donor 3:FIG. 3E-G).

Example 3

Detection of Systemic Immune Activation with IFN Inducible GenesExpression in Mouse PBMC by TLRL

Balb/c mice, 6-8 weeks of age, female, purchased from Vital River wereinjected intravenously with TLRL, at indicated time point, mice werebled and IFN inducible genes were examined by qPCR. Once pick time ofexpression IFN inducible genes was determined, a separated experimentwas performance with various dose of TLRL. At indicated time point, micewere bled and IFN inducible genes were examined. The QuantitativeReal-Time PCR was performed and gene expression data were normalizedrelative to geometric mean of two housekeeping genes (Actin):

Mouse Actin: F: (SEQ ID NO.: 1) CATTGCTGACAGGATGCAGAAGG, Mouse Actin R:(SEQ ID NO.: 2) TGCTGGAAGGTGGACAGTGAGG; Mouse Inf-b: F:  (SEQ ID NO.: 3)CTCCAGCACTGGGTGGAATG, Mouse Inf-b R:  (SEQ ID NO.: 4)AGTGGAGAGCAGTTGAGGAC; Mouse Mx2: F; (SEQ ID NO.: 5)GTGGCAGAGGGAGAATGTCG, Mouse Mx2 R: (SEQ ID NO.: 6)TAAAACAGCATAACCTTTTGCGA; Mouse Ifn-a: F: (SEQ ID NO.: 7)CCTGAGAGAGAAGAAACACAGCC, Mouse Ifn-a R: (SEQ ID NO.: 8)GGCTCTCCAGACTTCTGCTCTG; Mouse ISG15: F: (SEQ ID NO.: 9)CAGCAATGGCCTGGGACCTAA, Mouse ISG15R: (SEQ ID NO.: 10)GGAAAGCCGGCACACCAATC.

FIGS. 4A-4C depict expression of IFN inducible genes in mouse PBMC afterTLRL injection. RNA was isolated from PBMCs cryopreserved with TRIzolreagent at variable time points and Relative expression of IFN induciblegenes were determined by quantitative RT-PCR. MX2 gene was detected overtime course of 5 hours post TLRL injection (FIG. 4A) and MX2 and ISG15genes were measured with various dose of TLRL at 2 hours post injection(FIG. 4B and FIG. 4C). Values indicate the mRNA expression of indicatedIFN inducible genes relative to housekeeping gene Actin. Bar graphsrepresent data from 3 individual animals. **P<0.01; ***P<0.001.

Statistical Analysis

The significance of all comparisons was calculated using a Student'stwo-tailed t test assuming unequal variance between mock and samplegroups, and results considered significant when p<0.05. Correlationsbetween parameters were assessed using Spearman's rank correlation test,P values <0.05 were consider to be statistically significant.

Example 4

PD-L1 Expressed C26 Tumor Model

Combinational Efficacy of R848 and Anti-PD1 Antibody in Immune-CompetentMouse of PD-L1 Expressed C26 Tumor Model

The objective of this study was to determine the single or combinationalefficacy of R848 and anti-PD-1 monoclonal antibody in the mouse colonadenocarcinoma 26 (C26) tumor model in immune-competent mouse. C26 cellswere inoculated by subcutaneous injection into the right flank of BALB/cmice on Day 0. Tumors were monitored until they reached a mean tumorvolume of between 60 and 100 mm3. Mice were then administered over athree-week period 10 mg/kg IV (via tail vein) of rat anti-mouse PD-1monoclonal antibody alone twice weekly, 0.08 mg/kg of R848 alone onceweekly, or a combination of anti-PD-1 antibody and R848. Tumor volumeswere recorded twice weekly over the course of the study and the micewere observed regularly. Tumor-bearing mice (treatment, control) weresacrificed at Day 26 or if they became moribund before the tumor volumereached 2000 mm3. Limited inhibition in tumor growth was detectedfollowing treatment with R848 alone or anti-PD-1 antibody alone. Theantitumor activity of R848 alone or anti-PD-1 antibody alone wasmarkedly enhanced when given in combination. Following combinationtreatment with R848 and anti-PD-1 antibody, tumor size was greatlyreduced with 83.75% tumor inhibition. The differences in tumorinhibition by the combination treatment compared to that of eachcomponent is shown graphically (FIG. 5) and was highly significant(p=0.0002, anti-PD-1 and R848 combined versus R848 alone; p=0.0063anti-PD-1 and R848 combined versus anti-PD-1 alone). No adverse effectson clinical signs, body weight or survival were observed in animalsfollowing treatment with the combination of R848 and anti-PD-1 antibody.

Combinational Efficacy of Anti-PD-L1 and R848 in Immune-Competent Mouseof PD-L1 Expressed C26 Tumor Model (Study No. R-BDB001-0008)

The objective of this study was to determine the single or combinationalefficacy of R848 and anti-PD-L1 monoclonal antibody in the C26 tumormodel in immune-competent mouse. C26 cells were inoculated bysubcutaneous injection into the right flank of BALB/c mice on Day 0.Tumors were monitored until they reached a mean tumor volume of between60 and 100 mm3, inclusive. Mice were administered, over a 3-week period,10 mg/kg IV (via tail vein) of rat anti-mouse PD-L1 monoclonal antibodyalone twice weekly, 0.08 mg/kg of R848 alone once weekly, or acombination of anti-PD-L1 antibody and R848.

Limited inhibition in tumor growth was detected following treatment withR848 alone or anti-PD-L1 antibody alone. The antitumor activity of R848alone or anti-PD-L1 antibody alone was markedly enhanced when given incombination. Following combination treatment with R848 and anti-PD-L1antibody, tumor size was greatly reduced with 96% tumor inhibition. Thedifferences in tumor inhibition by the combination treatment compared tothat of each component is shown graphically (FIG. 6)) and was highlysignificant (p=0.0029, anti-PDL1 and R848 combined versus R848 alone;p=0.0074, anti-PDL1 and R848 combined versus anti-PDL1 alone). Noadverse effects on clinical signs, body weight or survival were observedin animals following treatment with the combination of R848 andanti-PD-L1 antibody.

1. A combination, comprising: (i) an effective amount of a PD-L/PD-1Axis antagonist antibody; and (ii) an effective amount of animmunotherapeutic, wherein the immunotherapeutic is4-amino-2-(ethoxymethyl)-a,a-di-methyl-1H-imidazo[4,5-c]quinoline-1-ethanol;wherein the PD-L/PD-1 Axis antagonist antibody and the immunotherapeuticare not covalently linked to each other.
 2. The combination of claim 1,wherein said PD-L/PD-1 Axis antagonist is selected from the groupconsisting of a PD-1 binding antagonist, a PD-L1 binding antagonist anda PD-L2 binding antagonist.
 3. The combination of claim 2, wherein thePD-L/PD-1 Axis antagonist is a PD-1 binding antagonist.
 4. Thecombination of claim 3, wherein the PD-1 binding antagonist is anantibody fragment.
 5. The combination of claim 5, wherein the PD-1binding antagonist is MDX-1106, Merck 3475, CT-011, AMP-224, or AMP-514.6. The combination of claim 2, wherein the PD-L/PD-1 Axis antagonist isa PD-L1 binding antagonist.
 7. The combination of claim 6, wherein thePD-L1 binding antagonist is an antibody.
 8. The combination of claim 7,wherein the PD-L1 binding antagonist is selected from the groupconsisting of: YW243.55.S70, MPDL3280A, MDX-1105, MEDI-4736, andMSB0010718C.
 9. The combination of claim 2, wherein the PD-L/PD-1 Axisantagonist is a PD-L2 binding antagonist.
 10. The combination of claim9, wherein the PD-L2 binding antagonist is an antibody.
 11. Thecombination of claim 9, wherein the PD-L2 binding antagonist is animmunoadhesin.
 12. The combination of any one of claim 1, wherein saidimmunotherapeutics is a compound of any one of formula (I) to (XIXb), ora pharmaceutically acceptable salt or solvate thereof.
 13. Thecombination of claim 1, further comprising an effective amount of an ananticancer agent.
 14. The combination of claim 13, wherein saidanticancer agent is an antimetabolite, an inhibitor of topoisomerase Iand II, an alkylating agent, a microtubule inhibitor, an antiandrogenagent, a GNRh modulator or mixtures thereof.
 15. The combination ofclaim 13, wherein said anticancer agent is a chemotherapeutic agentselected from the group consisting of tamoxifen, raloxifene,anastrozole, exemestane, letrozole, imatanib, paclitaxel,cyclophosphamide, lovastatin, minosine, gemcitabine, cytarabine,5-fluorouracil, methotrexate, docetaxel, goserelin, vincristine,vinblastine, nocodazole, teniposide etoposide, gemcitabine, epothilone,vinorelbine, camptothecin, daunorubicin, actinomycin D, mitoxantrone,acridine, doxorubicin, epirubicin, or idarubicin.
 16. The combination ofclaim 1, wherein said immunotherapeutic is of an amount that is capableof: (1) inducing IFN-α in an enriched human blood DCs; (2) inducingTNF-α in an enriched human blood DCs; (3) inducing IL-12-α in anenriched human blood DCs; (4) activating CD45+ immune cells in tumormicroenvironment; (5) activating CD4+ and CD8+ T cells in tumormicroenvironment; (6) activating NK cells in tumor microenvironment; (7)activating plasmacytoid dendritic cells (pDC) and myeloid dendriticcells (mDc) in tumor microenvironment; (8) activating macrophages andMonocytes in tumor microenvironment; and/or (9) increasing migratory DCsin draining lymph nodes.
 17. A kit, comprising the combination of claim1.